JAK inhibitors and uses thereof

ABSTRACT

The present invention provides methods for treating pruritus in a subject in need thereof comprising administering a therapeutically effective amount of a JAK inhibitor. Also provided are methods of intranasal administration of JAK inhibitors for use in treating pruritus.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of priority to PCT InternationalApplication No. PCT/US2017/018097 filed 16 Feb. 2017, which claims thebenefit of U.S. Provisional Application Ser. No. 62/295,875 filed on 16Feb. 2016, each of which is incorporated herein by reference in itsentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not Applicable.

MATERIAL INCORPORATED-BY-REFERENCE

The Sequence Listing, which is a part of the present disclosure,includes a computer readable form comprising nucleotide and/or aminoacid sequences of the present invention. The subject matter of theSequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present disclosure generally relates to methods for treatingpruritus, including chronic idiopathic pruritus, and pruritic componentsof other pruritic disorders as described herein, in a subject in needthereof comprising administering a therapeutically effective amount of aJAK inhibitor.

BACKGROUND OF THE INVENTION

Itchiness, i.e. the sensation that produces the desire to scratch, isclinically referred to as “pruritus.” There are multiple etiologies ofpruritus, and it is often a major clinical challenge to diagnose theunderlying etiology in order to adequately treat.

The multidimensional character of the itch experience is reflected inthe complexity of itch processing in the brain. Multiple neural pathwaysand molecular mechanisms responsible for producing the sensation of itchhave been identified, including histamine-dependent andhistamine-independent pathways. Ideally, effective treatment of prurituswould be informed by an understanding of the underlying etiology and theneuronal pathways involved in each specific disease. Although recentneurophysiological research has led to a better understanding of neuralpathways involved in itch response, the pathophysiology is still notcompletely understood. Accordingly, the state of the art does not allowa medical practitioner to predict whether a treatment will effectivelycontrol pruritus of different etiologies, particularly when the originof pruritus cannot be identified.

SUMMARY OF THE INVENTION

Among the various aspects of the present disclosure is the provision ofa method of treatment of pruritus using JAK inhibitors.

In some embodiments, the method includes administering to the subject atherapeutically effective amount of a JAK inhibitor.

In some embodiments, the subject is diagnosed with pruritus or chronicidiopathic pruritus and treatment prevents or reduces pruritus in thesubject.

In some embodiments, the subject has extremely severe itching or severeitching; or the subject has moderate or mild itching.

In some embodiments, the pruritus has lasted for at least at least sevenweeks; the pruritus has lasted for at least at least eight weeks; thepruritus has lasted for at least at least nine weeks; or the pruritushas lasted for at least at least ten weeks.

In some embodiments, the treatment reduces severity of itching in asubject, increases number of itch-free days in the subject, improvesquality of life of the subject, or any combination thereof.

In some embodiments, the subject is predisposed to pruritus or chronicidiopathic pruritus and treatment prevents a reoccurrence of chronicpruritus in the subject or reduces frequency of acute pruritus in thesubject.

In some embodiments, the JAK inhibitor is of formula (I),

or a pharmaceutically acceptable salt thereof, including all tautomersand stereoisomers thereof wherein:

U, V, W, X, Y, and Z, are selected from the group consisting of C and N;

R₁, R₂, R₃, or R₄ are independently selected from the group consistingof hydrogen; unsubstituted or substituted alkyl; unsubstituted orsubstituted alkenes; unsubstituted or substituted alkyne; acetamide;

and

-   -   R₁, R₂, R₃, or R₄ are optionally substituted with one or more        groups independently selected from the group consisting of        hydroxyl; hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀carboxyl,        straight chain or branched C₁₋₁₀alkyl, optionally containing        unsaturation; a C₂₋₆ cycloalkyl optionally containing        unsaturation or one oxygen or nitrogen atom; straight chain or        branched C₁₋₁₀alkyl amine; heterocyclyl; heterocyclic amine; and        aryl comprising a phenyl; heteroaryl containing from 1 to 4 N,        O, or S atoms; unsubstituted phenyl ring; substituted phenyl        ring; unsubstituted heterocyclyl; and substituted heterocyclyl;    -   the unsubstituted phenyl ring or substituted phenyl ring is        optionally substituted with one or more groups independently        selected from the group consisting of hydroxyl; C₁₋₁₀alkyl        hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀carboxyl; straight        chain or branched C₁₋₁₀alkyl, optionally containing        unsaturation; straight chain or branched C₁₋₁₀alkyl amine,        optionally containing unsaturation; a C₂₋₆ cycloalkyl optionally        containing unsaturation or one oxygen or nitrogen atom; straight        chain or branched C₁₋₁₀alkyl amine; heterocyclyl; heterocyclic        amine; aryl comprising a phenyl; and heteroaryl containing from        1 to 4 N, O, or S atoms; and    -   the unsubstituted heterocyclyl or substituted heterocyclyl is        optionally substituted with one or more groups independently        selected from the group consisting of hydroxyl; C₁₋₁₀alkyl        hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀carboxyl; straight        chain or branched C₁₋₁₀alkyl, optionally containing        unsaturation; straight chain or branched C₁₋₁₀alkyl amine,        optionally containing unsaturation; a C₂₋₆ cycloalkyl optionally        containing unsaturation or one oxygen or nitrogen atom;        heterocyclyl; straight chain or branched C₁₋₁₀alkyl amine;        heterocyclic amine; and aryl comprising a phenyl; and heteroaryl        containing from 1 to 4 N, O, or S atoms.

In some embodiments, the JAK inhibitor is selected from one or more ofthe group consisting of: tofacitinib, ruxolitinib, baricitinib,INCB039110, oclacitinib, AZD1480, fedratinib, AT9283, AG-490,momelotinib, WP1066, TG101209, gandotinib, NVP-BSK805, AZ 960,CEP-33779, Pacritinib, WHI-P154, XL019, S-Ruxolitinib, ZM 39923,Decernotinib, Cerdulatinib, filgotinib, FLLL32, BMS-911543, peficitinib,GLPG0634, GLPG0634 analogue, Go6976, curcumol, cucurbitacin,lestaurtinib, upadacitinib, CHZ868, Solcitinib (GSK 2586184), NS-018; ora derivative thereof; or pharmaceutically acceptable salt thereof.

In some embodiments, the JAK inhibitor is

In some embodiments, the JAK inhibitor is parenterally administered.

In some embodiments, the JAK inhibitor is administered intrathecally.

In some embodiments, the JAK inhibitor is administered intranasally.

In some embodiments, the JAK inhibitor is administered orally.

In some embodiments, the JAK inhibitor is administered on a daily basis.

In some embodiments, the JAK inhibitor is administered daily for atleast 7 consecutive days.

In some embodiments, the JAK inhibitor is administered daily for atleast 14 consecutive days.

In some embodiments, the JAK inhibitor is administered daily for atleast 30 consecutive days.

In some embodiments, the pruritus is a symptom of broad activation ofimmune responses or dysregulation of neuronal processes and sensoryperception.

In some embodiments, the subject is diagnosed with, or the pruritus is asymptom of, a disease or condition selected from one or more of thegroup consisting of: allergic reaction, arthropod bites, athlete's foot,atopic dermatitis (AD), atopic itch, atopic dermatitis-associated itch,autoimmune connective tissue disease, bacterial infection, biliary itch,broad activation of immune responses, body louse, bullous diseases,brachioradial pruritus, brain tumors, chronic idiopathic pruritus,contact dermatitis, cholestasis, cutaneous larva migrans, cutaneousT-cell lymphoma, nervous system damage, dandruff, delusionalparasitosis, dermatomyositis, dermatosis of pregnancy, diabetesmellitus, drug eruptions, dysregulation of neuronal processes andsensory perception, eczema, eosinophilic folliculitis, foreign objectsor devices on skin, fungal infection, gestational pemphigoid, head lice,herpes, hidradenitis suppurativa, hives, Hodgkin's disease,hyperparathyroidism, idiopathic chronic itch, inflammation, insectinfestation, insect bites, insect stings, intrahepatic cholestasis ofpregnancy, iron deficiency anemia, increased accumulation of exogenousopioids or synthetic opioids, internal cancer, jaundice, lichen planus,lichen sclerosus, lupus erythematosus, lymphoma, lymphoma—associateditch, leukemia-associated itch, malignancy, mastocytosis, menopause,multiple sclerosis, neoplasm, nerve irritation, neurogenic itch,neuropathic itch, notalgia paresthetica, notalgia obsessive-compulsivedisorders, paresthetica, parasitic infection, papular uritcaria,pediculosis, peripheral neuropathy, photodermatitis, polycythemia vera,psychiatric disease, psychogenic itch, pruritic popular eruption of HIV,pruritic urticarial papules and plaques of pregnancy (PUPPP), psoriasis,psoriasis-associated itch, psoriatic itch, pubic lice, punctatepalmoplantar keratoderma, renal itch, rheumatoid arthritis, scabies,scar growth, shaving, seborrheic dermatitis, stasis dermatitis, sunburn,swimmer's itch, systemic immune senescence, tactile hallucinations,Th17-associated inflammation, thyroid illness, uraemia, pruritus oruremic itch, urticaria, urticarial itch, varicella, viral infection,wound or scab healing, and xerosis.

In some embodiments, the pruritus has an inflammatory etiology; thepruritus has neuronal dysfunction etiology; or the pruritus has anunknown etiology.

In some embodiments, the JAK inhibitor is a TRPV1 inhibitor; modulatessignaling of IL-4 or IL-13 pathway; or targets the IL-4Rα signalingpathway.

In some embodiments, the method further includes administration of aTRPV1 inhibitor, dupilumab, or secukinumab.

Other objects and features will be in part apparent and in part pointedout hereinafter.

DESCRIPTION OF THE DRAWINGS

Those of skill in the art will understand that the drawings, describedbelow, are for illustrative purposes only. The drawings are not intendedto limit the scope of the present teachings in any way.

The application file contains at least one drawing executed in color.Copies of this patent application publication with color drawing(s) willbe provided by the Office upon request and payment of the necessary fee.

FIG. 1A-B depicts a representative photograph of (A) a subject withatopic dermatitis, and (B) a subject with CIP. Subjects with CIP lackthe overt skin inflammation seen in atopic dermatitis.

FIG. 2A-C depicts a representative photograph and histopathologicfindings on skin biopsy from a subject with CIP. (FIG. 2A) Clinicalphotograph of the patient described in Case 1 (see Example 1, TABLE 2).As is the case with many CIP patients, no distinct rash was appreciable.A skin biopsy was taken at the location depicted by the arrow. (FIG. 2B)and (FIG. 2C) Skin biopsy from the patient described in Case 4 (seeExample 1, TABLE 2) demonstrating (FIG. 2B) spongiosis with denselymphocytic infiltrates (arrows) on low power (scale bar represents 500μm), and (FIG. 2C) abundant eosinophils at high power (scale barrepresents 50 μm).

FIG. 3A-B depicts graphs illustrating that administration of a JAKinhibitor to subjects with CIP (n=5) improved their pruritus. (FIG. 3A)Average itch scores decreased about 50% following treatment with a JAKinhibitor for one month. Itch was assessed using the Numerical RatingScale (NRS). (FIG. 3B) Daily itch scores (y-axis; NRS) for a singlesubject from (FIG. 3A) over a 70 day period of time (x-axis). Thesubject reported no improvement in pruritus when treated with acyclosporine alone but reported significant improvement when switched toa JAK inhibitor (Xeljanz or tofacitinib).

FIG. 4 illustrates a model of JAK-mediated CIP. Multiple cytokines(TSLP, IL-4, IL-31) and other factors (e.g., serotonin, etc.) may act onsensory neurons to mediate itch. However, blockade of a single cytokinemay not be sufficient to abate itch. JAK inhibition at the neuronallevel may represent a therapeutic strategy to block multipleitch-activating pathways.

FIG. 5A-FIG. 5I shows type 2 cytokines directly activate itch-sensorypathways. (FIG. 5A) Representative gel of RT-PCR product of whole mousedorsal root ganglia (DRG), n=4. (FIG. 5B) Representative gel of RT-PCRproduct of whole human DRG, N=3. (FIG. 5C) Quantification of Il4ra,Il13ra1, and Il31ra by RT-qPCR in whole mouse DRG, n=4. (FIG. 5D)Representative calcium imaging trace of mouse DRG neurons in response torecombinant murine (rm)IL-4 and potassium chloride (KCl). (FIG. 5E)Representative calcium imaging trace of mouse DRG neurons in response tormIL-13 and KCl. (FIG. 5F) Representative calcium imaging trace of mouseDRG neurons in response to rmIL-31 and KCl. (FIG. 5G) rmIL-4-, rmIL-13-,rmIL-31-, and histamine-responsive neurons as a percentage of totalmouse DRG neurons, n>500 neurons. (FIG. 5H and FIG. 5I) Venn diagrams ofoverlapping responses of mouse DRG neurons to stimulation with (FIG. 5H)rmIL-4 or (FIG. 5I) rmIL-13 and subsequent rmIL-31 and histaminechallenge, n>300 neurons. Data are represented as mean±SEM. See alsoFIG. 12 and FIG. 13.

FIG. 6A-FIG. 6G shows chronic itch is associated with enhancedexpression of type 2 cytokine receptors in sensory ganglia. (FIG. 6A)Experimental schematic indicating daily topical treatment with vehiclecontrol (ethanol, EtOH) or MC903 to the ears of wild type (WT) mice.(FIG. 6B) Ear thickness measurements as percent change from baselineover the course of control or MC903 treatment, n∝4 per group. (FIG. 6C)Representative histopathology and (FIG. 6D) histology score of controland MC903-treated mice, n≥4 per group. (FIG. 6E) Scratching behavior ofcontrol and MC903-treated mice on Day 0, 4, 8, and 12, n≥4 per group.(FIG. 6F) Comparison of gene row Z-scores of regularized logarithmexpression values of select atopic dermatitis (AD)-associated genes inthe skin of control and MC903-treated mice, n=4 per group. (FIG. 6G)RT-qPCR for Il4ra, Il5ra, and Il13ra1 in sensory trigeminal ganglia fromcontrol and MC903-treated mice, n=8 per group. Scale bars indicate 100μm. Data are represented as mean±SEM. See also FIG. 14.

FIG. 7A-FIG. 7E shows type 2 immune cells interact with sensory nervefibers in the skin. (FIG. 7A) Experimental schematic indicating dailytopical treatment with vehicle control (EtOH) to one ear and MC903 tothe second ear of Na_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ mice. (FIG. 7B and FIG.7C) Representative images of intravital two-photon imaging of (FIG. 7B)control and (FIG. 7C) MC903-treated ears. Red arrows indicateNa_(v)1.8-tdTomato⁺ sensory fibers. Green arrows indicate IL-4-eGFP⁺cells. Representative stills from movies are provided in FIG. 16 andFIG. 17. (FIG. 7D) Instantaneous speed of a representative IL-4-eGFP⁺cell during its interactions with a sensory nerve fiber in MC903-treatedskin of a Na_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ mouse. The graph is split intothree components showing the speed of the cell as it approaches,interacts with, and then leaves the sensory nerve fiber. Stills from themovie of this cell track is provided in FIG. 18. (FIG. 7E) Mean speedover the duration of imaging of IL-4-eGFP⁺ cells in association withsensory nerve fibers compared to those not associated with nerve fibersin the MC903-treated skin of a Na_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ mouse,n>40 cells per group. Error bars represent SD. Scale bars indicate 100μm. See also FIG. 16-FIG. 18.

FIG. 8A-FIG. 8F shows neuronal IL-4Rα expression is necessary forchronic itch. (FIG. 8A) Experimental schematic indicating daily topicaltreatment with MC903 to the ears of IL-4Rα^(Δneuron) and littermatecontrol mice. (FIG. 8B) RT-qPCR of sensory trigeminal ganglia ofIL-4Rα^(Δneuron) mice, 7 per group. (FIG. 8C) Scratching behavior ofIL-4Rα^(Δneuron) mice compared to littermate controls over the course ofMC903 treatment, 7 per group. (FIG. 8D) Ear thickness measurements,(FIG. 8E) representative histopathology, and (FIG. 8F) histology scoreof MC903-treated IL-4Rα^(Δneuron) mice compared to littermate controls,n≥4 per group. Scale bars indicate 100 μm. Data are represented asmean±SEM.

FIG. 9A-FIG. 9O shows disruption of neuronal JAK1 reduces chronic itch.(FIG. 9A) Experimental schematic indicating twice daily intraperitoneal(i.p.) injection of control vehicle or high dose ruxolitinib (rux) (100μg) during MC903 treatment to the ears of wild type (WT) mice. (FIG. 9B)Scratching behavior, (FIG. 9C) ear thickness measurement, (FIG. 9D)representative histopathology, and (FIG. 9E) histology score of i.p.control and rux-treated mice on Day 7, n≥10 mice per group. (FIG. 9F)Experimental schematic indicating single intrathecal (i.t.) injection ofcontrol vehicle or low dose rux (10 μg) 24 hours before behavioralassays of MC903-treated WT mice. (FIG. 9G) Scratching behavior, (FIG.9H) ear thickness measurement, (FIG. 9I) representative histopathology,and (FIG. 9J) histology score of i.t. control and rux-treated mice onDay 7, n 10 mice per group. (FIG. 9K) Experimental schematic indicatingMC903 treatment of JAK1^(Δneuron) and littermate control mice. (FIG. 9L)Scratching behavior, (FIG. 9M) ear thickness measurement, (FIG. 9N)representative histopathology, and (FIG. 9O) histology score ofJAK1^(Δneuron) mice compared to littermate controls on Day 7, n≥8 pergroup. Scale bars indicate 100 μm. Data are represented as mean±SEM. Seealso FIG. 15.

FIG. 10A-FIG. 10H shows CIP is a distinct chronic itch disorder thatexhibits neuronal dysregulation and severe itch despite minimal skininflammation. (FIG. 10A and FIG. 10B) Representative gross clinicalpictures of (FIG. 10A) atopic dermatitis (AD) and (FIG. 10B) chronicidiopathic pruritus (CIP). (FIG. 100 and FIG. 10D) Representativehistopathology images of (FIG. 100) AD and (FIG. 10D) CIP. (FIG. 10E)Histology score of AD and CIP patient biopsies, N≥4 per group. (FIG.10F) Numerical Rating Scale (NRS) itch scores of AD and CIP patients,N≥22 per group. (FIG. 10G) Clustering of AD, CIP, and healthy controlskin samples by gene row Z-scores of regularized logarithm expressionvalues of the top 100 differentially expressed genes in AD versushealthy control skin. (FIG. 10H) Gene set enrichment analysis (GSEA) ofa direct comparison between CIP and AD skin. Positive enrichment scoresindicate enrichment in CIP. Scale bars indicate 100 μm. Data arerepresented as mean±SEM. See also TABLE 3 and TABLE 4.

FIG. 11A-FIG. 11B shows patients with refractory CIP improve whentreated with JAK inhibitors. (FIG. 11A) NRS itch scores for a cohort ofCIP patients (N=5) given the JAK inhibitor tofacitinib. Each pointrepresents a patient before and after treatment with tofacitinib.Significance was calculated using the paired t-test. Error barsrepresent SD. (FIG. 11B) Daily NRS itch scores of two CIP patientstreated with tofacitinib including one patient treated with cyclosporineimmediately preceding treatment with tofacitinib (black). See also TABLE5.

FIG. 12 shows IL-5 does not activate DRG neurons. Relates to FIG. 5.Representative calcium imaging traces of mouse DRG neurons challengedwith recombinant murine IL-5, histamine (Hist), capsaicin (Cap), andpotassium chloride (KCl), n>300 neurons.

FIG. 13 shows predicted pruriceptive pathways are enriched in neuronsexpressing type 2 cytokine receptors compared to other predicted sensorymodalities. Relates to FIG. 5. Expression profile within mouse DRGneurons at the single cell level of cluster-defining and selecteditch-related genes for predicted sensory modalities. Numbers in heat mapindicate fraction of positive cells by thresholding method. Full datasetand methods available in Usoskin et al., 2015.

FIG. 14 shows MC903 treatment results in inflammatory changes in theskin also observed in human atopic dermatitis (AD). Relates to FIG. 6.Comparison of gene row Z-scores of regularized logarithm expressionvalue of the top 100 differentially expressed genes in the skin ofMC903-treated mice compared to controls determined by p-value. Genes arelisted based on increasing adjusted p-value.

FIG. 15 shows predicted itch-sensory neurons express JAK signalingcomponents. Relates to FIG. 9. Expression profile within mouse DRGneurons at the single cell level of the JAK family of proteins forpredicted sensory modalities. Numbers in heat map indicate fraction ofpositive cells by thresholding method. Full dataset and methodsavailable in Usoskin et al., 2015.

FIG. 16 are stills of a movie showing control treatment results in fewtype 2 immune cells in the skin. Relates to FIG. 7. Representativetime-lapse movie of intravital two-photon imaging of aNa_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ control ear. Movie shows few IL-4-eGFP⁺cells in the ear skin.

FIG. 17 are stills of a movie showing MC903 treatment results in manytype 2 immune cells near sensory nerve fibers in the skin. Relates toFIG. 6. Representative time-lapse movie of intravital two-photon imagingof a Na_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ MC903-treated ear. Movie shows manyIL-4-eGFP⁺ cells near sensory nerve fibers in the ear skin.

FIG. 18 are stills of a movie showing type 2 immune cells interact withsensory nerve fibers in the skin. Relates to FIG. 7. Representativetime-lapse movie of intravital two-photon imaging of aNa_(v)1.8-tdTomato⁺ IL-4-eGFP⁺ MC903-treated ear demonstrating anexample track of an IL-4-eGFP⁺ cell approaching a sensory nerve fiber,interacting with it, then leaving the area of the fiber.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure is based, at least in part, on the discovery thatimmune defense mechanisms directly modulate host behavior; type 2 immunecells interact with sensory nerve fibers in the skin; sensoryneuron-specific deletion of IL-4Rα or JAK1 reduces chronic itch; andclinical studies demonstrate that JAK inhibitors relieve chronic itch.As shown herein, the present disclosure provides methods for treatingchronic idiopathic pruritus (CIP) in a subject in need thereof.

In particular, until now, treatment of CIP has not been successful. Asdescribed herein is a solution to an unmet need of treating CIP.Furthermore, successful treatment using an intranasal administration ofJAK inhibitors has been shown herein. Intranasal administration isclinically advantageous because of the ease of use and increase inpatient compliance.

In contrast to many inflammatory pruritic diseases, which are typicallyresponsive to both topical and systemic anti-inflammatory drugs, chronicidiopathic pruritus is often resistant to many different types ofanti-inflammatory treatments. Surprisingly, applicants have discoveredthat JAK inhibitors, which can also be anti-inflammatory compounds, canbe used to effectively treat chronic idiopathic pruritus. Additionalaspects of the invention are described in further detail below.

Mammals have evolved neurophysiologic reflexes such as coughing andscratching to expel invading pathogens and noxious environmentalfactors. It is well established that these responses are also associatedwith chronic inflammatory diseases such as asthma and atopic dermatitis.However, the mechanisms by which inflammatory pathways promotesensations such as itch remain poorly understood.

As described herein, type 2 cytokines, IL-4 and IL-13, directlystimulate sensory neurons and that chronic itch is dependent on neuronalIL-4Rα and JAK1 signaling. As described herein, patients withrecalcitrant chronic itch markedly improve when treated with JAKinhibitors. Thus, signaling mechanisms previously ascribed to the immunesystem may represent novel therapeutic targets within the nervoussystem. Collectively, these studies reveal an evolutionarily conservedparadigm in which the sensory nervous system employs classical immunesignaling pathways to influence mammalian behavior.

As described herein, it was demonstrated that sensory neurons aredirectly activated by the classical immune signaling molecules IL-4 andIL-13 along previously defined itch-sensory pathways, provoking thehypothesis that neuronal type 2 cytokine signaling mediates chronicitch. Indeed, employing sensory neuron-specific genetic deletion ofIL-4Rα, it was found that neuronal expression of IL-4Rα is required forthe development of chronic itch in an established mouse model of atopicdermatitis (AD) associated with robust itch. Based on IL-4Rα signalingbiology, it was thus hypothesized that dysregulated neuronal Januskinase (JAK) signaling may be a conserved mechanism by which chronicitch is mediated. Strikingly, both pharmacologic JAK inhibition andsensory neuron-specific genetic deletion of JAK1 demonstrated abatementof chronic itch. Although JAK inhibitors are well-established asanti-inflammatory agents (Hirahara et al., 2016; Schwartz et al., 2016),whether they exhibit additional neuromodulatory properties is not known.In support of this possibility, it was observed dramatic improvement ofitch symptoms in a small cohort of CIP patients treated off-label withthe JAK inhibitor tofacitinib after having failed other broadanti-inflammatory therapies. Thus, these signaling mechanisms previouslyascribed to the immune system may also represent novel targets withinthe sensory nervous system for the treatment of chronic itch. Beyonditch, the discovery of these novel neuroimmunologic pathways includingIL-4Rα and JAK1 signaling may reveal new insights into sensoryperception at multiple barrier surfaces and how these pathways modulatehost behavior. It is important to note that many cytokines beyond IL-4and IL-13, even neurotransmitters (e.g., serotonin), have been shown tosignal through JAKs. Thus, the mechanism of action of JAK inhibition islikely to extend beyond type 2 cytokine-associated itch such as atopicdermatitis or CIP and to many other itch disorders.

JAK Inhibitor

Another aspect of the present disclosure provides a JAK inhibitor. A“JAK inhibitor” or “Janus kinase inhibitor” can refer to apharmaceutically active ingredient that functions by inhibiting theactivity of one or more enzymes of the Janus kinase family (e.g. JAK1,JAK2, JAK3, TYK2), thereby interfering with the JAK-STAT signalingpathway. In some embodiments, a JAK inhibitor inhibits JAK1. In someembodiments, a JAK inhibitor inhibits JAK2. In some embodiments, a JAKinhibitor inhibits JAK3. In some embodiments, a JAK inhibitor inhibitsTYK2. In some embodiments, a JAK inhibitor inhibits JAK1 and JAK2. Insome embodiments, a JAK inhibitor inhibits JAK2 and JAK3. In someembodiments, a JAK inhibitor inhibits JAK1 and JAK3. In someembodiments, a JAK inhibitor inhibits JAK1 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK2 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK3 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK1, JAK2 and JAK3. In someembodiments, a JAK inhibitor inhibits JAK1, JAK2 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK1, JAK3 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK2, JAK3 and TYK2. In someembodiments, a JAK inhibitor inhibits JAK1, JAK2, JAK3 and TYK2.Non-limiting examples of suitable JAK inhibitors may include itacitinib(INCB039110), baricitinib, cucurbitacin (JSI124), fedratinib, filgotinib(GLPG0634), gandotinib (LY2784544), lestaurtinib, momelotinib (CYT387),pacritinib, peficitinib, ruxolitinib, tofacitinib, upadacitinib,AZD1480, BMS911543, CHZ868, solcitinib (GSK2586184), NS-018, and XL-019.In exemplary embodiments, a JAK inhibitor is chosen from ruxolitinib andtofacitinib.

A “JAK inhibitor” or “Janus kinase inhibitor” can be a pharmaceuticallyactive ingredient that functions by inhibiting the activity of one ormore enzyme of the Janus kinase family (e.g. JAK1, JAK2, JAK3, TYK2),thereby interfering with the JAK-STAT signaling pathway.

In some embodiments, a JAK inhibitor can be based on

a benizimidazole core,

a purine core,

or

a pyrrolo pyridine core,

In some embodiments, a JAK inhibitor can be of formula (I),

wherein U, V, W, X, Y, and Z, are selected from the group consisting ofC and N.

In some embodiments, R₁, R₂, R₃, or R₄ can be hydrogen, alkyl (e.g.,methyl), or acetamide;

In some embodiments, R₁, R₂, R₃, or R₄ can optionally substituted withone or more groups independently selected from the group consisting ofhydroxyl; C₁₋₁₀alkyl hydroxyl; amine; C₁₋₁₀carboxylic acid;C₁₋₁₀carboxyl, straight chain or branched C₁₋₁₀alkyl, optionallycontaining unsaturation; a C₂₋₆ cycloalkyl optionally containingunsaturation or one oxygen or nitrogen atom; straight chain or branchedC₁₋₁₀alkyl amine; heterocyclyl; heterocyclic amine; and aryl comprisinga phenyl; heteroaryl containing from 1 to 4 N, O, or S atoms;unsubstituted phenyl ring; substituted phenyl ring; unsubstitutedheterocyclyl; and substituted heterocyclyl;

-   -   the unsubstituted phenyl ring or substituted phenyl ring is        optionally substituted with one or more groups independently        selected from the group consisting of hydroxyl; C₁₋₁₀alkyl        hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀carboxyl, straight        chain or branched C₁₋₁₀alkyl, optionally containing        unsaturation; straight chain or branched C₁₋₁₀alkyl amine,        optionally containing unsaturation; a C₂₋₆ cycloalkyl optionally        containing unsaturation or one oxygen or nitrogen atom; straight        chain or branched C₁₋₁₀alkyl amine; heterocyclyl; heterocyclic        amine; aryl comprising a phenyl; and heteroaryl containing from        1 to 4 N, O, or S atoms; and    -   the unsubstituted heterocyclyl or substituted heterocyclyl is        optionally substituted with one or more groups independently        selected from the group consisting of hydroxyl; C₁₋₁₀alkyl        hydroxyl; amine; C₁₋₁₀carboxylic acid; C₁₋₁₀carboxyl, straight        chain or branched C₁₋₁₀alkyl, optionally containing        unsaturation; straight chain or branched C₁₋₁₀alkyl amine,        optionally containing unsaturation; a C₂₋₆ cycloalkyl optionally        containing unsaturation or one oxygen or nitrogen atom;        heterocyclyl; straight chain or branched C₁₋₁₀alkyl amine;        heterocyclic amine; and aryl comprising a phenyl; and heteroaryl        containing from 1 to 4 N, O, or S atoms.

In some embodiments, a JAK inhibitor can be tofacitinib,

In some embodiments, a JAK inhibitor can be ruxolitinib,

In some embodiments, a JAK inhibitor can be baricitinib,

In some embodiments, a JAK inhibitor can be itacitinib (INCB039110),

In some embodiments, a JAK inhibitor can be oclacitinib,

In some embodiments, a JAK inhibitor can be AZD1480,

In some embodiments, a JAK inhibitor can be fedratinib,

In some embodiments, a JAK inhibitor can be AT9283,

In some embodiments, a JAK inhibitor can be AG-490,

In some embodiments, a JAK inhibitor can be momelotinib,

In some embodiments, a JAK inhibitor can be WP1066,

In some embodiments, a JAK inhibitor can be TG101209,

In some embodiments, a JAK inhibitor can be gandotinib,

In some embodiments, a JAK inhibitor can be NVP-BSK805 2HCl,

In some embodiments, a JAK inhibitor can be AZ 960,

In some embodiments, a JAK inhibitor can be CEP-33779,

In some embodiments, a JAK inhibitor can be pacritinib

In some embodiments, a JAK inhibitor can be WHI-P154

In some embodiments, a JAK inhibitor can be XL019,

In some embodiments, a JAK inhibitor can be S-Ruxolitinib

In some embodiments, a JAK inhibitor can be ZM 39923 HCl,

In some embodiments, a JAK inhibitor can be Decernotinib,

In some embodiments, a JAK inhibitor can be Cerdulatinib,

In some embodiments, a JAK inhibitor can be filgotinib,

In some embodiments, a JAK inhibitor can be FLLL32,

In some embodiments, a JAK inhibitor can be BMS-911543,

In some embodiments, a JAK inhibitor can be peficitinib,

In some embodiments, a JAK inhibitor can be GLPG0634 analogue,

In some embodiments, a JAK inhibitor can be Go6976,

In some embodiments, a JAK inhibitor can be Curcumol,

In some embodiments, a JAK inhibitor can be cucurbitacin,

In some embodiments, a JAK inhibitor can be lestaurtinib,

In some embodiments, a JAK inhibitor can be upadacitinib,

In some embodiments, a JAK inhibitor can be CHZ868,

In some embodiments, a JAK inhibitor can be Solcitinib (GSK 2586184),

In some embodiments, a JAK inhibitor can be NS-018,

The term “imine” or “imino”, as used herein, unless otherwise indicated,includes a functional group or chemical compound containing acarbon-nitrogen double bond. The expression “imino compound”, as usedherein, unless otherwise indicated, refers to a compound that includesan “imine” or an “imino” group as defined herein.

The term “hydroxyl”, as used herein, unless otherwise indicated,includes —OH.

The terms “halogen” and “halo”, as used herein, unless otherwiseindicated, include a chlorine, chloro, Cl; fluorine, fluoro, F; bromine,bromo, Br; or iodine, iodo, or I.

The term “aryl”, as used herein, unless otherwise indicated, include acarbocyclic aromatic group. Examples of aryl groups include, but are notlimited to, phenyl, benzyl, naphthyl, or anthracenyl.

The terms “amine” and “amino”, as used herein, unless otherwiseindicated, include a functional group that contains a nitrogen atom witha lone pair of electrons and wherein one or more hydrogen atoms havebeen replaced by a substituent such as, but not limited to, an alkylgroup or an aryl group.

The term “alkyl”, as used herein, unless otherwise indicated, includessaturated monovalent hydrocarbon radicals having straight or branchedmoieties, such as but not limited to, methyl, ethyl, propyl, butyl,pentyl, hexyl, octyl groups, etc. Representative straight-chain loweralkyl groups include, but are not limited to, -methyl, -ethyl,-n-propyl, -n-butyl, -n-pentyl, -n-hexyl, -n-heptyl and -n-octyl; whilebranched lower alkyl groups include, but are not limited to, -isopropyl,-sec-butyl, -isobutyl, -tert-butyl, -isopentyl, 2-methylbutyl,2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, 3,3-dimethylpentyl,2,3,4-trimethylpentyl, 3-methylhexyl, 2,2-dimethylhexyl,2,4-dimethylhexyl, 2,5-dimethylhexyl, 3,5-dimethylhexyl,2,4-dimethylpentyl, 2-methylheptyl, 3-methylheptyl, unsaturated C₁-C₈alkyls include, but are not limited to, -vinyl, -allyl, -1-butenyl,-2-butenyl, -isobutylenyl, -1-pentenyl, -2-pentenyl,-3-methyl-1-butenyl, -2-methyl-2-butenyl, -2,3-dimethyl-2-butenyl,1-hexyl, 2-hexyl, 3-hexyl, -acetylenyl, -propynyl, -1-butynyl,-2-butynyl, -1-pentynyl, -2-pentynyl, or -3-methyl-1 butynyl. An alkylcan be saturated, partially saturated, or unsaturated.

The term “carboxyl”, as used herein, unless otherwise indicated,includes a functional group consisting of a carbon atom double bonded toan oxygen atom and single bonded to a hydroxyl group (—COOH).

The term “alkenyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon double bond whereinalkyl is as defined above and including E and Z isomers of said alkenylmoiety. An alkenyl can be partially saturated or unsaturated.

The term “alkynyl”, as used herein, unless otherwise indicated, includesalkyl moieties having at least one carbon-carbon triple bond whereinalkyl is as defined above. An alkynyl can be partially saturated orunsaturated.

The term “acyl”, as used herein, unless otherwise indicated, includes afunctional group derived from an aliphatic carboxylic acid, by removalof the hydroxyl (—OH) group.

The term “alkoxyl”, as used herein, unless otherwise indicated, includesO-alkyl groups wherein alkyl is as defined above and O representsoxygen. Representative alkoxyl groups include, but are not limited to,—O-methyl, —O-ethyl, —O-n-propyl, —O-n-butyl, —O-n-pentyl, —O-n-hexyl,—O-n-heptyl, —O-n-octyl, —O-isopropyl, —O-sec-butyl, —O-isobutyl,—O-tert-butyl, —O-isopentyl, —O-2-methylbutyl, —O-2-methylpentyl,—O-3-methylpentyl, —O-2,2-dimethylbutyl, —O-2,3-dimethylbutyl,—O-2,2-dimethylpentyl, —O-2,3-dimethylpentyl, —O-3,3-dimethylpentyl,—O-2,3,4-trimethylpentyl, —O-3-methylhexyl, —O-2,2-dimethylhexyl,—O-2,4-dimethylhexyl, —O-2,5-dimethylhexyl, —O-3,5-dimethylhexyl,—O-2,4dimethylpentyl, —O-2-methylheptyl, —O-3-methylheptyl, —O-vinyl,—O-allyl, —O-1-butenyl, —O-2-butenyl, —O-isobutylenyl, —O-1-pentenyl,—O-2-pentenyl, —O-3-methyl-1-butenyl, —O-2-methyl-2-butenyl,—O-2,3-dimethyl-2-butenyl, —O-1-hexyl, —O-2-hexyl, —O-3-hexyl,—O-acetylenyl, —O-propynyl, —O-1-butynyl, —O-2-butynyl, —O-1-pentynyl,—O-2-pentynyl and —O-3-methyl-1-butynyl, —O-cyclopropyl, —O-cyclobutyl,—O-cyclopentyl, —O-cyclohexyl, —O-cycloheptyl, —O-cyclooctyl,—O-cyclononyl and —O-cyclodecyl, —O—CH₂-cyclopropyl, —O—CH₂-cyclobutyl,—O—CH₂-cyclopentyl, —O—CH₂-cyclohexyl, —O—CH₂-cycloheptyl,—O—CH₂-cyclooctyl, —O—CH₂-cyclononyl, —O—CH₂-cyclodecyl,—O—(CH₂)₂-cyclopropyl, —O—(CH₂)₂-cyclobutyl, —O—(CH₂)₂-cyclopentyl,—O—(CH₂)₂-cyclohexyl, —O—(CH₂)₂-cycloheptyl, —O—(CH₂)₂-cyclooctyl,—O—(CH₂)₂-cyclononyl, or —O—(CH₂)₂-cyclodecyl. An alkoxyl can besaturated, partially saturated, or unsaturated.

The term “cycloalkyl”, as used herein, unless otherwise indicated,includes a non-aromatic, saturated, partially saturated, or unsaturated,monocyclic or fused, spiro or unfused bicyclic or tricyclic hydrocarbonreferred to herein containing a total of from 3 to 10 carbon atoms,preferably 3 to 8 ring carbon atoms. Examples of cycloalkyls include,but are not limited to, C₃-C₈ cycloalkyl groups include, but are notlimited to, -cyclopropyl, -cyclobutyl, -cyclopentyl, -cyclopentadienyl,-cyclohexyl, -cyclohexenyl, -1,3-cyclohexadienyl, -1,4-cyclohexadienyl,-cycloheptyl, -1,3-cycloheptadienyl, -1,3,5-cycloheptatrienyl,-cyclooctyl, and -cyclooctadienyl.

The term “cycloalkyl” also includes -lower alkyl-cycloalkyl, whereinlower alkyl and cycloalkyl are as defined herein. Examples of -loweralkyl-cycloalkyl groups include, but are not limited to,—CH₂-cyclopropyl, —CH₂-cyclobutyl, —CH₂-cyclopentyl,—CH₂-cyclopentadienyl, —CH₂-cyclohexyl, —CH₂-cycloheptyl, or—CH₂-cyclooctyl.

The term “heterocyclic”, as used herein, unless otherwise indicated,includes an aromatic or non-aromatic cycloalkyl in which one to four ofthe ring carbon atoms are independently replaced with a heteroatom fromthe group consisting of 0, S and N. Representative examples of aheterocycle include, but are not limited to, benzofuranyl,benzothiophene, indolyl, benzopyrazolyl, coumarinyl, isoquinolinyl,pyrrolyl, pyrrolidinyl, thiophenyl, furanyl, thiazolyl, imidazolyl,pyrazolyl, triazolyl, quinolinyl, pyrimidinyl, pyridinyl, pyridonyl,pyrazinyl, pyridazinyl, isothiazolyl, isoxazolyl, (1,4)-dioxane,(1,3)-dioxolane, 4,5-dihydro-1H-imidazolyl, or tetrazolyl. Heterocyclescan be substituted or unsubstituted. Heterocycles can also be bonded atany ring atom (i.e., at any carbon atom or heteroatom of theheterocyclic ring). A heterocyclic can be saturated, partiallysaturated, or unsaturated.

The term “cyano”, as used herein, unless otherwise indicated, includes a—CN group.

The term “alcohol”, as used herein, unless otherwise indicated, includesa compound in which the hydroxyl functional group (—OH) is bound to acarbon atom. In particular, this carbon center should be saturated,having single bonds to three other atoms.

When carbocyclyl and heterocyclyl are substituted, they are typicallysubstituted by 1 or 2 substituents (e.g. 1 substituent). Typically thesubstituent is methyl. More typically carbocyclyl and heterocyclylgroups are unsubstituted.

When aryl and heteroaryl are substituted, they are typically substitutedby 1, 2 or 3 (e.g. 1 or 2) substituents. Substituents for aryl andheteroaryl are selected from C₁₋₆alkyl (e.g. methyl), C₂₋₆alkenyl (e.g.buten-3-yl), C₂₋₆alkynyl (e.g. butyn-3-yl), C₁₋₆haloalkyl (e.g.fluoromethyl, trifluoromethyl), —C₁₋₆thioalkyl (e.g. —S-methyl),—SOC₁₋₄alkyl (e.g. —SOmethyl), —SO₂C₁₋₄alkyl (e.g. —SO₂methyl),C₁₋₆alkoxy- (e.g. methoxy, ethoxy), —O—C₃₋₈cycloalkyl (e.g.—O-cyclopentyl), C₃₋₈cycloalkyl (e.g. cyclopropyl, cyclohexyl),—SO₂C₃₋₈cycloalkyl (e.g. —SO₂cyclohexyl), —SOC₃₋₆cycloalkyl (e.g.—SOcyclopropyl), C₃₋₆alkenyloxy- (e.g. —O-buten-2-yl), C₃₋₆alkynyloxy-(e.g. —O-buten-2-yl), —C(O)C₁₋₆alkyl (e.g. —C(O)ethyl), —C(O)OC₁₋₆alkyl(e.g. —C(O)O-methyl), C₁₋₆alkoxy-C₁₋₆alkyl- (e.g. methoxy-ethyl-),nitro, halogen (e.g. fluoro, chloro, bromo), cyano, hydroxyl, —C(O)OH,—NH₂, —NHC₁₋₄alkyl (e.g. —NHmethyl), —N(C₁₋₄alkyl)(C₁₋₄alkyl) (e.g.—N(methyl)₂), —C(O)N(C₁₋₄alkyl)(C₁₋₄alkyl) (e.g. —C(O)N(methyl)₂),—C(O)NH₂, —C(O)NH(C₁₋₄alkyl) (e.g. —C(O)NHmethyl),—C(O)NH(C₃₋₁₀cycloalkyl) (e.g. —C(O)NHcyclopropyl). More typically,substituents will be selected from C₁₋₆alkyl (e.g. methyl),C₁₋₆haloalkyl (e.g. C₁₋₆fluoroalkyl, e.g. CF₃), C₁₋₆alkoxy (e.g. OMe),halogen and hydroxy.

When an R group represents heteroaryl, examples include monocyclic (e.g.5 and 6 membered) and bicyclic (e.g. 9 and 10 membered, particularly 9membered) heteroaryl rings, especially rings containing nitrogen atoms(e.g. 1 or 2 nitrogen atoms). A suitable bicyclic heteroaryl ring is a9-membered heteroaryl ring containing 1 or 2 nitrogen atoms, especiallya benzene ring fused to a 5-membered ring containing one or two nitrogenatoms (e.g. 1H-benzoimidazolyl). Most suitably the point of attachmentis through a benzene ring, e.g. the group is 1H-benzoimidazol-5-yl.Aforementioned heteroaryl groups may either be unsubstituted (which ismore typical) or may suitably be substituted by one or more (e.g. 1 or2) substituents selected from alkyl (e.g. C₁₋₄ alkyl such as Me),alkoxy- (e.g. C₁₋₄ alkoxy- such as OMe) and halogen (e.g. F).

When an R group represents —C₃₋₈carbocyclyl-heteroaryl, examples ofcarbocyclyl include cycloalkyl (e.g. cyclohexyl) and cycloalkenyl (e.g.cyclohexenyl), examples of heteroaryl groups include monocyclic (e.g. 5or 6 membered, particularly 5 membered) rings especially ringscontaining nitrogen atoms e.g. 1 or 2 nitrogen atoms. Aforementionedheteroaryl groups may either be unsubstituted (which is more typical) ormay suitably be substituted by one or more (e.g. 1 or 2) substituentsselected from alkyl (e.g. C₁₋₄ alkyl such as Me), alkoxy- (e.g. C₁₋₄alkoxy- such as OMe) and halogen (e.g. F). A suitable heteroaryl groupis imidazol-1-yl. An exemplary —C₃₋₈carbocyclyl-heteroaryl group is3-imidazol-1-yl-cyclohexyl-.

When an R group represents —C₂₋₆alkenyheteroaryl, examples of C₂₋₆alkenyl include C₂₋₄ alkenyl, in particular propenyl and examples ofheteroaryl groups include monocyclic (e.g. 5 or 6 membered, particularly5 membered) rings especially rings containing nitrogen atoms e.g. 1 or 2nitrogen atoms. Aforementioned heteroaryl groups may either beunsubstituted (which is more typical) or may suitably be substituted byone or more (e.g. 1 or 2) substituents selected from alkyl (e.g.C₁₋₄alkyl such as Me), alkoxy- (e.g. C₁₋₄ alkoxy- such as OMe) andhalogen (e.g. F). A suitable heteroaryl group is imidazolyl,particularly imidazol-1-yl. An exemplary -alkenylheteroaryl group is3-imidazol-1-yl-prop-2-enyl-.

When an R group represents —C₁₋₆alkylheteroaryl, examples of C₁₋₆ alkylinclude C₁₋₅alkyl or C₁₋₄alkyl, especially C₂₋₅alkyl or C₂₋₄ alkyl, inparticular propyl, and examples of heteroaryl groups include monocyclic(e.g. 5 or 6 membered, particularly 5 membered) rings especially ringscontaining nitrogen atoms e.g. 1 or 2 nitrogen atoms. Aforementionedheteroaryl groups may either be unsubstituted (which is most typical) ormay suitably be substituted by one or more (e.g. 1 or 2) substituentsselected from alkyl (e.g. C₁₋₄ alkyl such as Me), alkoxy- (e.g. C₁₋₄alkoxy- such as OMe) and halogen (e.g. F). A suitable heteroaryl groupis imidazol-1-yl. A particularly suitable -alkylheteroaryl group is3-imidazol-1-yl-propyl-.

When R represents —C₁₋₆alkylheteroaryl, examples wherein alkyl isbranched include:

When an R group represents (CH₂)_(a)CR⁵R⁶(CH₂)_(b)heteroaryl wherein aand b independently represent integers 0-5 provided that a+b=0-5 and R⁵and R⁶ are alkylene which together with the carbon to which they areattached form a C₃-C₅ cycloalkyl group, examples include:

Particular examples of R heteroaryl groups include a 5-membered ringcontaining 2 or 3 nitrogen atoms, which ring may optionally besubstituted (e.g. in particular by one or two groups, such as methyl,for example:

Other examples of R heteroaryl groups include a 9-membered bicyclic ringcontaining 2 nitrogen atoms, which ring may optionally be substituted,for example:

Clearly, the heteroaryl groups shown above may also be present as partof a larger R function such as —C₃₋₈carbocyclyl-heteroaryl,—C₂₋₆alkenylheteroaryl or —C₁₋₆alkylheteroaryl.

When an R group represents —C₁₋₈alkyl, examples include methyl, ethyl,propyl (e.g. n-propyl, isopropyl), butyl (e.g. n-butyl-sec-butyl,isobutyl and tert-butyl), pentyl (e.g. n-pentyl, 3,3,-dimethylpropyl),hexyl, heptyl and octyl.

When an R group represents optionally substituted aryl, aryl maytypically represent phenyl. Exemplary substituted phenyl groups include3-methylphenyl-, 2,3-dichlorophenyl-, 2,3-difluorophenyl-,2,4-dichlorophenyl-, 2,4-difluororophenyl-, 2,4-dimethoxyphenyl-,2,4-dimethylphenyl-, 2,4-bis(trifluoromethyl)phenyl-,2,4,6-trifluorophenyl-, 2,4,6-trimethylphenyl-, 2,6-dichlorophenyl-,2,6-difluorophenyl-, 2,6-dimethoxyphenyl-,2,6-difluoro-4-(methoxy)phenyl-, 2-isopropyl-6-methylphenyl-,3-(cyclopentyloxy)-4-methoxyphenyl-, 3,4,5-trimethoxyphenyl-,3,4-dimethoxyphenyl-, 3,4-dichlorophenyl-, 3,4-difluorophenyl-,3,4-dimethylphenyl-, 3,4,5-trifluorophenyl-,3,5-bis(trifluororomethyl)phenyl-, 3,5-dimethoxyphenyl-,2-methoxyphenyl-, 3-methoxyphenyl-, 4-(trifluoromethyl)phenyl-,4-bromo-2-(trifluoromethyl)phenyl-, 4-bromophenyl-,4-chloro-3-(trifluoromethyl)phenyl-, 4-chlorophenyl-, 4-cyanophenyl-,4-ethoxyphenyl-, 4-ethylphenyl-, 4-fluorophenyl-, 4-isopropylphenyl-,4-methoxyphenyl-, 4-ethoxyphenyl-, 4-propoxyphenyl-, 4-butoxyphenyl-,4-pentoxyphenyl-, 4-isopropyloxyphenyl-, 4-tetrafluoroethyloxyphenyl-.Alternatively, R² may represent unsubstituted phenyl-. Further exemplarysubstituted phenyl groups include 2,3,4-trifluorophenyl,2,3-difluoro-4-methylphenyl, 2-bromo-4-fluorophenyl-,2-bromo-5-fluorophenyl-, 2-chlorophenyl-, 2-fluorophenyl-,2-fluoro-5-(trifluoromethyl)phenyl-, 2-hydroxy-3-methoxyphenyl-,2-hydroxy-5-methylphenyl-, 3-chlorophenyl-, 3-fluorophenyl-,3-fluoro-4-(trifluoromethyl)phenyl-,3-fluoro-5-(trifluoromethyl)phenyl-,2-fluoro-4-(trifluoromethyl)phenyl-, 3-fluoro-4-(methoxy)phenyl-,3-hydroxy-4-methoxyphenyl-, 4-bromo-2-fluorophenyl,4-chloro-3-(trifluoromethyl)phenyl-, 4-chloro-3-methylphenyl,4-chlorophenyl-, 4-fluorophenyl- and 4-propoxyphenyl-.

When an R group represents optionally substituted aryl and arylrepresents naphthyl, examples include unsubstituted naphthyl (e.g.naphthalen-1-yl, naphthalen-2-yl, naphthalen-3-yl) as well assubstituted naphthyl (e.g. 4-methyl-naphthalen-2-yl-,5-methyl-naphthalen-3-yl-, 7-methyl-naphthalen-3-y- and4-fluoro-naphthalen-2-yl-).

When an R group represents optionally substituted heteroaryl, examplesinclude monocyclic rings (e.g. 5 or 6 membered rings) and bicyclic rings(e.g. 9 or 10 membered rings) which may optionally be substituted.Example 5 membered rings include pyrrolyl (e.g. pyrrol-2-yl) andimidazolyl (e.g. 1H-imidazol-2-yl or 1H-imidazol-4-yl), pyrazolyl (e.g.1H-pyrazol-3-yl), furanyl (e.g. furan-2-yl), thiazolyl (e.g.thiazol-2-yl), thiophenyl (e.g. thiophen-2-yl, thiophen-3-yl). Example 6membered rings include pyridinyl (e.g. pyridin-2-yl and pyridin-4-yl).Specific substituents that may be mentioned are one or more e.g. 1, 2 or3 groups selected from halogen, hydroxyl, alkyl (e.g. methyl) andalkoxy- (e.g. methoxy-). Example substituted 5 membered rings include4,5-dimethyl-furan-2-yl-, 5-hydroxymethyl-furan-2-yl-,5-methyl-furan-2-yl- and 6-methyl-pyridin-2-yl-. An example substituted6-membered ring is 1-oxy-pyridin-4-yl-. Example 9 membered rings include1H-indolyl (e.g. 1H-indol-3-yl, 1H-indol-5-yl), benzothiophenyl (e.g.benzo[b]thiophen-3-yl, particularly 2-benzo[b]thiophen-3-yl),benzo[1,2,5]-oxadiazolyl(e.g. benzo[1,2,5]-oxadiazol-5-yl),benzo[1,2,5]-thiadiazolyl(e.g. benzo[1,2,5]-thiadiazol-5-yl,benzo[1,2,5]thiadiazol-6-yl). Example 10 membered rings includequinolinyl (e.g. quinolin-3-yl, quinolin-4-yl, quinolin-8-yl). Specificsubstituents that may be mentioned are one or more e.g. 1, 2 or 3 groupsselected from halogen, hydroxyl, alkyl (e.g. methyl) and alkoxy- (e.g.methoxy-). Example substituted 9-membered rings include1-methyl-1H-indol-3-yl, 2-methyl-1H-indol-3-yl, 6-methyl-1H-indol-3-yl.Example substituted 10 membered rings include 2-chloro-quinolin-3-yl,8-hydroxy-quinolin-2-yl, oxo-chromenyl (e.g. 4-oxo-4H-chromen-3-yl) and6-methyl-4-oxo-4H-chromen-3-yl.

When an R group represents carbocyclyl, examples include cycloalkyl andcycloalkenyl. Examples of cycloalkyl include cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl and cycloheptyl. Examples of cycloalkenylinclude cyclohexenyl (e.g. cyclohex-2-enyl, cyclohex-3-enyl). Examplesof substituted carbocyclyl include 2-methyl-cyclohexyl-,3-methyl-cyclohexyl-, 4-methyl-cyclohexyl-, 2-methyl-cyclohex-2-enyl,2-methyl-cyclohex-3-enyl, 3-methyl-cyclohex-3-enyl,3-methyl-cyclohex-3-enyl.

When an R group represents heterocyclyl (which may optionally besubstituted), examples include tetrahydrofuranyl, morpholinyl,piperdinyl, 3,4-dihydro-2H-pyranyl, pyrrolidinyl,methyltetrahydrofuranyl- (e.g. 5-methyltetrahydrofuran-2-yl-).

When an R group represents —C₁₋₄alkylaryl, examples include-alkyl(substituted phenyl) e.g. in which phenyl is substituted by one ormore groups selected from alkyl, fluoroalkyl, halogen and alkoxy (e.g.methyl, trifluoromethyl, tert-butyl, chloro, fluoro and methoxy) and,for example, alkyl is C₁₋₄ alkyl. Another specific group is-alkyl(bicyclic aryl) e.g. wherein bicyclic aryl is optionallysubstituted naphthyl. A further specific group is benzyl.

When an R group represents —C₁₋₄alkylheteroaryl in which heteroaryl isoptionally substituted, examples include methylheteroaryl and-ethylheteroaryl (e.g. 1-heteroarylethyl- and 2-heteroarylethyl-),-propylheteroaryl and -butylheteroaryl in which heteroaryl is optionallysubstituted. Specific examples of -alkylheteroaryl groups includepyridinylmethyl-, N-methyl-pyrrol-2-methyl-N-methyl-pyrrol-2-ethyl-,N-methyl-pyrrol-3-methyl-, N-methyl-pyrrol-3-ethyl-,2-methyl-pyrrol-1-methyl-, 2-methyl-pyrrol-1-ethyl-,3-methyl-pyrrol-1-methyl-, 3-methyl-pyrrol-1-ethyl-, 4-pyridino-methyl-,4-pyridino-ethyl-, 2-(thiazol-2-yl)-ethyl-, 2-ethyl-indol-1-methyl-,2-ethyl-indol-1-ethyl-, 3-ethyl-indol-1-methyl-, 3-ethyl-indol-1-ethyl-,4-methyl-pyridin-2-methyl-, 4-methyl-pyridin-2-yl-ethyl-,4-methyl-pyridin-3-methyl-, 4-methyl-pyridin-3-ethyl-.

When an R group represents —C₁₋₄alkyl-carbocyclyl (which may optionallybe substituted), examples include -methyl-cyclopentyl,-methyl-cyclohexyl, -ethyl-cyclohexyl, -propyl-cyclohexyl,-methyl-cyclohexenyl, -ethyl-cyclohexenyl, -methyl(4-methylcyclohexyl)and -propyl (3-methylcyclyohexyl).

When an R group represents —C₁₋₄alkylheterocyclyl(which may optionallybe substituted); examples include -methyl-tetrahydrofuranyl (e.g.-methyl-tetrahydrofuran-2-yl, -methyl-tetrahydrofuran-3-yl),-ethyl-tetrahydrofuranyl, -methyl-piperidinyl.

When an R group represents phenyl substituted by phenyl or phenylsubstituted by a monocyclic heteroaryl group, in which any of aforesaidphenyl and heteroaryl groups may optionally be substituted, typicallythe phenyl ring connected directly to the nitrogen atom is unsubstitutedand the terminal phenyl ring or the monocyclic heteroaryl ring isoptionally substituted by one, two or three substitutents (e.g. one ortwo, e.g. one). Typically the terminal phenyl or monocyclic heteroarylgroup is unsubstituted. Typically the terminal phenyl or monocyclicheteroaryl group substitutes the other phenyl group at the 4-position.

When an R group represents phenyl substituted by phenyl in which any ofaforesaid phenyl groups may optionally be substituted, examples include-biphenyl-4-yl.

When an R group represents phenyl substituted by a monocyclic heteroarylgroup, in which any of aforesaid phenyl and heteroaryl groups mayoptionally be substituted, examples include 4-(oxazol-5-yl)phenyl-.

When an R group represents phenyl substituted by benzyloxy in which anyof aforesaid phenyl and benzyloxy groups may optionally be substituted,examples include 4-benzyloxy-phenyl-, 4-(3-methylbenzyloxy)phenyl- and4-(4-methylbenzyloxy)phenyl-.

When an R group represents optionally substituted phenyl fused tooptionally substituted carbocyclyl, examples include indanyl (e.g.indan-4-yl-, 2-methyl-indan-4-yl-), indenyl and tetralinyl.

When an R group represents optionally substituted phenyl fused tooptionally substituted heterocyclyl, examples includebenzo[1,3]dioxo-4-yl- and 2,3-dihydro-benzo[1,4]dioxin-4-yl-.

When an R group represents —C₁₋₄alkyl(phenyl substituted by phenyl),examples include biphenyl-4-yl-methyl-.

When an R group represents —C₁₋₄alkyl(phenyl substituted by a monocyclicheteroaryl group), examples include 4-(oxazol-5-yl)phenyl-methyl-.

When an R group represents —C₁₋₄alkyl(phenyl substituted by benzyloxy)in which any of aforesaid phenyl and benzyloxy groups may optionally besubstituted, examples include 4-benzyloxy-phenyl-methyl-,4-(3-methylbenzyloxy)phenyl-methyl- and4-(4-methylbenzyloxy)phenyl-methyl-.

When an R group represents —C₁₋₄alkyl(optionally substituted phenylfused to optionally substituted carbocyclyl), examples includeindanyl-methyl- (e.g. indan-4-yl-methyl-, 2-methyl-indan-4-yl-methyl-),indenyl-methyl- and tetralinyl-methyl-.

When an R group represents —C₁₋₄alkyl(optionally substituted phenylfused to optionally substituted heterocyclyl); examples includebenzo[1,3]dioxo-4-yl-methyl- and2,3-dihydro-benzo[1,4]dioxin-4-yl-methyl-.

When an R group represents —C₁₋₄alkyl, examples include methyl, ethyl,propyl (e.g. n-propyl, isopropyl) and butyl (e.g. n-butyl-sec-butyl,isobutyl and tert-butyl).

When an R group represents optionally substituted aryl, aryl maytypically represent phenyl. Exemplary substituted phenyl groups include2,4-dichlorophenyl-, 2,4-difluororophenyl-, 2,4-dimethoxyphenyl-,2,4-dimethylphenyl-, 2,4-bis(trifluoromethyl)phenyl-,2,4,6-trifluorophenyl-, 2,4,6-trimethylphenyl-, 2,6-dichlorophenyl-,2,6-difluorophenyl-, 2,6-dimethoxyphenyl-, 2-isopropyl-6-methylphenyl-,3-(cyclopentyloxy)-4-methoxyphenyl-, 3,4,5-trimethoxyphenyl-,3,4-dimethoxyphenyl-, 3,4-dichlorophenyl-, 3,4-dimethylphenyl-,3,4,5-trifluorophenyl-, 3,5-bis(trifluororomethyl)phenyl-,3,5-dimethoxyphenyl-, 3-methoxyphenyl-, 4-(trifluoromethyl)phenyl-,4-bromo-2-(trifluoromethyl)phenyl-, 4-bromophenyl-,4-chloro-3-(trifluoromethyl)phenyl-, 4-chlorophenyl-, 4-cyanophenyl-,4-ethoxyphenyl-, 4-ethylphenyl-, 4-fluorophenyl-, 4-isopropylphenyl-,4-methoxyphenyl-. Alternatively, R³ may represents unsubstitutedphenyl-. Further exemplary substituted phenyl groups include2-bromo-4-fluorophenyl-, 2-bromo-5-fluorophenyl-, 2-chlorophenyl-,2-fluoro-5-(trifluoromethyl)phenyl-, 2-hydroxy-3-methoxyphenyl-,2-hydroxy-5-methylphenyl-, 3-chlorophenyl-,3-fluoro-4-(trifluoromethyl)phenyl-, 3-hydroxy-4-methoxyphenyl-,4-chloro-3-(trifluoromethyl)phenyl-, 4-chlorophenyl-, 4-fluorophenyl-and 4-propoxyphenyl-.

When R¹ and R² or R³ and R⁴ are joined to form a carbocyclyl ring, whichis optionally substituted by one or more C₁₋₂alkyl groups, examplesinclude cycloalkyl (e.g. cyclopropyl, cyclopentyl and cyclohexyl) andcycloalkenyl (e.g. cyclohexenyl).

When R¹ and R² or R³ and R⁴ are joined to form a carbocyclyl ring whichis fused to phenyl; examples include indanyl (e.g. indan-2-yl) andtetralinyl.

When R¹ and R² or R³ and R⁴ are joined to form a carbocyclyl ring whichis fused to monocyclic heteroaryl; examples include 5-memberedcarbocyclyl fused to 6-membered heteroaryl, 6-membered carbocyclyl fusedto 6-membered heteroaryl, 5-membered carbocyclyl fused to 5-memberedheteroaryl and 6-membered carbocyclyl fused to 5-membered heteroaryl.The monocyclic heteroaryl to which carbocyclyl is fused contains atleast one heteroatom (e.g. one, two or three heteroatoms, e.g. one ortwo, e.g. one heteroatom).

When an R group represents —C₁₋₈alkyl examples include methyl, ethyl,propyl (e.g. n-propyl, isopropyl), butyl (e.g. n-butyl-sec-butyl,isobutyl and tert-butyl), pentyl (e.g. n-pentyl, 3,3,-dimethylpropyl),hexyl, heptyl and octyl.

When an R group represents —C(O)C₁₋₆alkyl; examples include—C(O)C₁₋₄alkyl such as —C(O)methyl, —C(O)ethyl, —C(O)propyl and—C(O)butyl.

The term “solvate” is intended to mean a solvate form of a specifiedcompound that retains the effectiveness of such compound. Examples ofsolvates include compounds of the invention in combination with, forexample: water, isopropanol, ethanol, methanol, dimethylsulfoxide(DMSO), ethyl acetate, acetic acid, or ethanolamine.

The term “mmol”, as used herein, is intended to mean millimole. The term“equiv”, as used herein, is intended to mean equivalent. The term “mL”,as used herein, is intended to mean milliliter. The term “g”, as usedherein, is intended to mean gram. The term “kg”, as used herein, isintended to mean kilogram. The term “μg”, as used herein, is intended tomean micrograms. The term “h”, as used herein, is intended to mean hour.The term “min”, as used herein, is intended to mean minute. The term“M”, as used herein, is intended to mean molar. The term “μL”, as usedherein, is intended to mean microliter. The term “μM”, as used herein,is intended to mean micromolar. The term “nM”, as used herein, isintended to mean nanomolar. The term “N”, as used herein, is intended tomean normal. The term “amu”, as used herein, is intended to mean atomicmass unit. The term “° C.”, as used herein, is intended to mean degreeCelsius. The term “wt/wt”, as used herein, is intended to meanweight/weight. The term “v/v”, as used herein, is intended to meanvolume/volume. The term “MS”, as used herein, is intended to mean massspectroscopy. The term “HPLC”, as used herein, is intended to mean highperformance liquid chromatograph. The term “RT”, as used herein, isintended to mean room temperature. The term “e.g.”, as used herein, isintended to mean example. The term “N/A”, as used herein, is intended tomean not tested.

As used herein, the expression “pharmaceutically acceptable salt” refersto pharmaceutically acceptable organic or inorganic salts of a compoundof the invention. Preferred salts include, but are not limited, tosulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate,bisulfate, phosphate, acid phosphate, isonicotinate, lactate,salicylate, acid citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate,or pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the parent compound. Furthermore, a pharmaceuticallyacceptable salt may have more than one charged atom in its structure.Instances where multiple charged atoms are part of the pharmaceuticallyacceptable salt can have multiple counterions. Hence, a pharmaceuticallyacceptable salt can have one or more charged atoms and/or one or morecounterion. As used herein, the expression “pharmaceutically acceptablesolvate” refers to an association of one or more solvent molecules and acompound of the invention. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine. As used herein, the expression “pharmaceuticallyacceptable hydrate” refers to a compound of the invention, or a saltthereof, that further includes a stoichiometric or non-stoichiometricamount of water bound by non-covalent intermolecular forces.

Pruritus and Conditions Associated with Pruritus

JAK inhibitors, as described herein, can treat pruritus and conditionsassociated with pruritus as a symptom. Pruritus can be referred to asthe sensation that produces the desire to scratch. As described herein,it is shown that patients with recalcitrant chronic itch markedlyimprove when treated with JAK inhibitors.

As used herein, the terms “pruritus” and “itch” can be interchangeable.

TABLE 1, below, provides a summary of common diseases or conditionsassociated with pruritus as a major symptom. When the origin of prurituscannot be identified (i.e., is not due to a known disease, see e.g.,TABLE 1), the condition is referred to as idiopathic pruritus.

TABLE 1 Common diseases with pruritus as a major symptom GENERALCATEGORY SPECIFIC DISEASE Infestations, bites Scabies Arthropod bitesand stings Pediculosis Inflammation Atopic dermatitis Papular uritcariaStasis dermatitis Drug eruptions Allergic > irritant contact Bullousdiseases dermatitis Mastocytosis Seborrheic dermatitis EosinophilicPsoriasis folliculitis Lichen planus Pruritic popular Urticaria eruptionof HIV Autoimmune Scabies Scabies connective tissue Lichen sclerosusLupus disease Dermatomyositis erythematosus Infection Bacterial FungalViral Parasitic Neoplastic Cutaneous T-cell lymphoma Polycythemia vera

Chronic Pruritus and Chronic Idiopathic Pruritus

“Chronic pruritus” can be pruritus lasting longer than six weeks.Chronic idiopathic pruritus” or “CIP” refers to itch of unknown originlasting greater than six weeks. Because the itch is of unknown origin, asubject with chronic idiopathic pruritus does not have a disease listedin TABLE 1. Subjects diagnosed with chronic idiopathic pruritus do nothave the overt skin inflammation seen in other inflammatory pruriticdiseases, such as atopic dermatitis. The initial evaluation of a patientwho has chronic idiopathic pruritus typically includes a complete bloodcount with a differential count, a chest radiograph, and tests ofhepatic, renal, and thyroid function. In many cases, subjects diagnosedwith chronic idiopathic pruritus have pruritus despite two or moretreatments over a period of at least six weeks. Examples of treatmentsmay include, but are not limited to, the use of mild cleaners,emollients, topical anesthetics, coolants, antihistamines,anticonvulsants, antidepressants, μ-opioid antagonists, neuroactivemedications (e.g. gabapentin, pregabalin, etc.), cortitcosteroid, andphototherapy.

Conditions associated with pruritus, treatable by JAK inhibitors, can bea disease or conditions wherein a symptom is pruritus. For example, adisease or condition associated with chronic idiopathic pruritus can beany of those diseases or conditions known by a person of ordinary skillin the art as being associated with the pruritus. As used herein, theterms “symptom” and “clinical sign” can be interchangeable. Symptoms caninclude those observable or measureable conditions or behaviors that aremeasured in known or established diagnostic assessments. For example,diagnostic assessments for a determination of pruritus can be made by aNumerical Rating Scale (NRS) score, Visual Analog Scale (VAS) score, oran Itch-Free Days (IFD) score, or by a scoring system such as theEppendorf Itch Questionnaire or the Patient Benefit Index, Version forPatients with Pruritus (PBI-P), or the 5-D Itch Scale. Non-limitingexamples of some symptoms of chronic idiopathic pruritus, that may beused in such assessments or scoring systems, include: quality of life oritching (e.g., distribution, duration, degree, improvement/worsening,impact on sleep, leisure, social, housework, errands, work/school,etc.). Itching may be reported as ranging from extremely severe (e.g.,9.0-10.0 using the VAS), to severe (e.g., 7.0-8.9 using the VAS), tomoderate (e.g., 3.0-6.9 using the VAS), to mild (e.g.; 0.1-2.9 using theVAS).

One aspect of the present disclosure provides a subject in need oftreatment for chronic idiopathic pruritus (CIP). In some embodiments, asubject in need of treatment for CIP is a subject diagnosed with CIP.Subjects diagnosed with CIP have pruritus of unknown origin that haslasted for at least six weeks. For example, the pruritus has lasted forat least six, at least seven, at least eight, at least nine, at leastten, at least eleven, or at least twelve weeks. The subject may haveextremely severe itching, severe itching, moderate itching or milditching. In other embodiments, a subject in need of treatment for CIP isa subject predisposed to CIP. For example, a subject may be predisposedto CIP if the subject was previously treated for CIP and the prurituswas resolved. The subject may have had extremely severe itching, severeitching, moderate itching or mild itching. Contemplated herein is thetreatment of subjects of any age.

In some of the above embodiments, a subject in need of treatment for CIPmay have immunologic deficits associated with Th2 polarizationincluding, but not limited to, elevated serum IgE levels, peripheraleosinophilia, tissue eosinophil infiltration, CD8 lympohpenia, or anycombination thereof. The subject may be ≥50 years of age, ≥55 years ofage, ≥60 years of age, or ≥65 years of age. Alternatively, the subjectmay be ≤50 years of age.

Conditions associated with pruritus and treatable with JAK inhibitorscan include, but are not limited to allergic reaction, arthropod bites,athlete's foot, atopic dermatitis (AD), atopic itch, atopicdermatitis-associated itch, autoimmune connective tissue disease,bacterial infection, biliary itch, broad activation of immune responses,body louse, bullous diseases, brachioradial pruritus, brain tumors,chronic idiopathic pruritus, contact dermatitis, cholestasis, cutaneouslarva migrans, cutaneous T-cell lymphoma, damage of the nervous system,dandruff, delusional parasitosis, dermatomyositis, dermatosis ofpregnancy, diabetes mellitus, drug eruptions, dysregulation of neuronalprocesses and sensory perception, eczema, eosinophilic folliculitis,foreign objects or devices on skin, fungal infection, gestationalpemphigoid, head lice, herpes, hidradenitis suppurativa, hives,Hodgkin's disease, hyperparathyroidism, idiopathic chronic itch,inflammation, insect infestation, insect bites, insect stings,intrahepatic cholestasis of pregnancy, iron deficiency anemia, increasedaccumulation of exogenous opioids or synthetic opioids, internal cancer,jaundice, lichen planus, lichen sclerosus, lupus erythematosus,lymphoma, lymphoma—associated itch, leukemia-associated itch,malignancy, mastocytosis, menopause, multiple sclerosis, neoplasm, nerveirritation, neurogenic itch, neuropathic itch, notalgia paresthetica,notalgia obsessive-compulsive disorders, paresthetica, parasiticinfection, papular uritcaria, pediculosis, peripheral neuropathy,photodermatitis, polycythemia vera, psychiatric disease, psychogenicitch, pruritic popular eruption of HIV, pruritic urticarial papules andplaques of pregnancy (PUPPP), psoriasis, psoriasis-associated itch,psoriatic itch, pubic lice, punctate palmoplantar keratoderma, renalitch, rheumatoid arthritis, scabies, scar growth, shaving, seborrheicdermatitis, stasis dermatitis, sunburn, swimmer's itch, systemic immunesenescence, tactile hallucinations, Th17-associated inflammation,thyroid illness, uraemia, pruritus or uremic itch, urticaria, urticarialitch, varicella, viral infection, wound or scab healing, or xerosis.

Molecular Engineering

The following definitions and methods are provided to better define thepresent invention and to guide those of ordinary skill in the art in thepractice of the present invention. Unless otherwise noted, terms are tobe understood according to conventional usage by those of ordinary skillin the relevant art.

The terms “heterologous DNA sequence”, “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

Expression vector, expression construct, plasmid, or recombinant DNAconstruct is generally understood to refer to a nucleic acid that hasbeen generated via human intervention, including by recombinant means ordirect chemical synthesis, with a series of specified nucleic acidelements that permit transcription or translation of a particularnucleic acid in, for example, a host cell. The expression vector can bepart of a plasmid, virus, or nucleic acid fragment. Typically, theexpression vector can include a nucleic acid to be transcribed operablylinked to a promoter.

A “promoter” is generally understood as a nucleic acid control sequencethat directs transcription of a nucleic acid. An inducible promoter isgenerally understood as a promoter that mediates transcription of anoperably linked gene in response to a particular stimulus. A promotercan include necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter can optionally include distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription.

A “transcribable nucleic acid molecule” as used herein refers to anynucleic acid molecule capable of being transcribed into a RNA molecule.Methods are known for introducing constructs into a cell in such amanner that the transcribable nucleic acid molecule is transcribed intoa functional mRNA molecule that is translated and therefore expressed asa protein product. Constructs may also be constructed to be capable ofexpressing antisense RNA molecules, in order to inhibit translation of aspecific RNA molecule of interest. For the practice of the presentdisclosure, conventional compositions and methods for preparing andusing constructs and host cells are well known to one skilled in the art(see e.g., Sambrook and Russel (2006) Condensed Protocols from MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in MolecularBiology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook andRussel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., ColdSpring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk,C. P. 1988. Methods in Enzymology 167, 747-754).

The “transcription start site” or “initiation site” is the positionsurrounding the first nucleotide that is part of the transcribedsequence, which is also defined as position +1. With respect to thissite all other sequences of the gene and its controlling regions can benumbered. Downstream sequences (i.e., further protein encoding sequencesin the 3′ direction) can be denominated positive, while upstreamsequences (mostly of the controlling regions in the 5′ direction) aredenominated negative.

“Operably-linked” or “functionally linked” refers preferably to theassociation of nucleic acid sequences on a single nucleic acid fragmentso that the function of one is affected by the other. For example, aregulatory DNA sequence is said to be “operably linked to” or“associated with” a DNA sequence that codes for an RNA or a polypeptideif the two sequences are situated such that the regulatory DNA sequenceaffects expression of the coding DNA sequence (i.e., that the codingsequence or functional RNA is under the transcriptional control of thepromoter). Coding sequences can be operably-linked to regulatorysequences in sense or antisense orientation. The two nucleic acidmolecules may be part of a single contiguous nucleic acid molecule andmay be adjacent. For example, a promoter is operably linked to a gene ofinterest if the promoter regulates or mediates transcription of the geneof interest in a cell.

A “construct” is generally understood as any recombinant nucleic acidmolecule such as a plasmid, cosmid, virus, autonomously replicatingnucleic acid molecule, phage, or linear or circular single-stranded ordouble-stranded DNA or RNA nucleic acid molecule, derived from anysource, capable of genomic integration or autonomous replication,comprising a nucleic acid molecule where one or more nucleic acidmolecule has been operably linked.

A constructs of the present disclosure can contain a promoter operablylinked to a transcribable nucleic acid molecule operably linked to a 3′transcription termination nucleic acid molecule. In addition, constructscan include but are not limited to additional regulatory nucleic acidmolecules from, e.g., the 3′-untranslated region (3′ UTR). Constructscan include but are not limited to the 5′ untranslated regions (5′ UTR)of an mRNA nucleic acid molecule which can play an important role intranslation initiation and can also be a genetic component in anexpression construct. These additional upstream and downstreamregulatory nucleic acid molecules may be derived from a source that isnative or heterologous with respect to the other elements present on thepromoter construct.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.

“Transformed,” “transgenic,” and “recombinant” refer to a host cell ororganism such as a bacterium, cyanobacterium, animal or a plant intowhich a heterologous nucleic acid molecule has been introduced. Thenucleic acid molecule can be stably integrated into the genome asgenerally known in the art and disclosed (Sambrook 1989; Innis 1995;Gelfand 1995; Innis & Gelfand 1999). Known methods of PCR include, butare not limited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Theterm “untransformed” refers to normal cells that have not been throughthe transformation process.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

Design, generation, and testing of the variant nucleotides, and theirencoded polypeptides, having the above required percent identities andretaining a required activity of the expressed protein is within theskill of the art. For example, directed evolution and rapid isolation ofmutants can be according to methods described in references including,but not limited to, Link et al. (2007) Nature Reviews 5(9), 680-688;Sanger et al. (1991) Gene 97(1), 119-123; Ghadessy et al. (2001) ProcNatl Acad Sci USA 98(8) 4552-4557. Thus, one skilled in the art couldgenerate a large number of nucleotide and/or polypeptide variantshaving, for example, at least 95-99% identity to the reference sequencedescribed herein and screen such for desired phenotypes according tomethods routine in the art.

Nucleotide and/or amino acid sequence identity percent (%) is understoodas the percentage of nucleotide or amino acid residues that areidentical with nucleotide or amino acid residues in a candidate sequencein comparison to a reference sequence when the two sequences arealigned. To determine percent identity, sequences are aligned and ifnecessary, gaps are introduced to achieve the maximum percent sequenceidentity. Sequence alignment procedures to determine percent identityare well known to those of skill in the art. Often publicly availablecomputer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR)software is used to align sequences. Those skilled in the art candetermine appropriate parameters for measuring alignment, including anyalgorithms needed to achieve maximal alignment over the full-length ofthe sequences being compared. When sequences are aligned, the percentsequence identity of a given sequence A to, with, or against a givensequence B (which can alternatively be phrased as a given sequence Athat has or comprises a certain percent sequence identity to, with, oragainst a given sequence B) can be calculated as: percent sequenceidentity=X/Y100, where X is the number of residues scored as identicalmatches by the sequence alignment program's or algorithm's alignment ofA and B and Y is the total number of residues in B. If the length ofsequence A is not equal to the length of sequence B, the percentsequence identity of A to B will not equal the percent sequence identityof B to A.

Generally, conservative substitutions can be made at any position solong as the required activity is retained. So-called conservativeexchanges can be carried out in which the amino acid which is replacedhas a similar property as the original amino acid, for example theexchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser byThr. For example, amino acids with similar properties can be Aliphaticamino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine);Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine,Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids(e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine,Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); orAcidic and their Amide (e.g., Aspartate, Glutamate, Asparagine,Glutamine). Deletion is the replacement of an amino acid by a directbond. Positions for deletions include the termini of a polypeptide andlinkages between individual protein domains. Insertions areintroductions of amino acids into the polypeptide chain, a direct bondformally being replaced by one or more amino acids. Amino acid sequencecan be modulated with the help of art-known computer simulation programsthat can produce a polypeptide with, for example, improved activity oraltered regulation. On the basis of this artificially generatedpolypeptide sequences, a corresponding nucleic acid molecule coding forsuch a modulated polypeptide can be synthesized in-vitro using thespecific codon-usage of the desired host cell.

“Highly stringent hybridization conditions” are defined as hybridizationat 65° C. in a 6×SSC buffer (i.e., 0.9 M sodium chloride and 0.09 Msodium citrate). Given these conditions, a determination can be made asto whether a given set of sequences will hybridize by calculating themelting temperature (T_(m)) of a DNA duplex between the two sequences.If a particular duplex has a melting temperature lower than 65° C. inthe salt conditions of a 6×SSC, then the two sequences will nothybridize. On the other hand, if the melting temperature is above 65° C.in the same salt conditions, then the sequences will hybridize. Ingeneral, the melting temperature for any hybridized DNA:DNA sequence canbe determined using the following formula: T_(m)=81.5°C.+16.6(log₁₀[Na⁺])+0.41(fraction G/C content)−0.63(%formamide)−(600/l). Furthermore, the T_(m) of a DNA:DNA hybrid isdecreased by 1-1.5° C. for every 1% decrease in nucleotide identity (seee.g., Sambrook and Russel, 2006).

Host cells can be transformed using a variety of standard techniquesknown to the art (see, e.g., Sambrook and Russel (2006) CondensedProtocols from Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002)Short Protocols in Molecular Biology, 5th ed., Current Protocols,ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: ALaboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10:0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167,747-754). Such techniques include, but are not limited to, viralinfection, calcium phosphate transfection, liposome-mediatedtransfection, microprojectile-mediated delivery, receptor-mediateduptake, cell fusion, electroporation, and the like. The transfectedcells can be selected and propagated to provide recombinant host cellsthat comprise the expression vector stably integrated in the host cellgenome.

Exemplary nucleic acids which may be introduced to a host cell include,for example, DNA sequences or genes from another species, or even genesor sequences which originate with or are present in the same species,but are incorporated into recipient cells by genetic engineeringmethods. The term “exogenous” is also intended to refer to genes thatare not normally present in the cell being transformed, or perhapssimply not present in the form, structure, etc., as found in thetransforming DNA segment or gene, or genes which are normally presentand that one desires to express in a manner that differs from thenatural expression pattern, e.g., to over-express. Thus, the term“exogenous” gene or DNA is intended to refer to any gene or DNA segmentthat is introduced into a recipient cell, regardless of whether asimilar gene may already be present in such a cell. The type of DNAincluded in the exogenous DNA can include DNA which is already presentin the cell, DNA from another individual of the same type of organism,DNA from a different organism, or a DNA generated externally, such as aDNA sequence containing an antisense message of a gene, or a DNAsequence encoding a synthetic or modified version of a gene.

Host strains developed according to the approaches described herein canbe evaluated by a number of means known in the art (see e.g., Studier(2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005)Production of Recombinant Proteins: Novel Microbial and EukaryoticExpression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004)Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Methods of down-regulation or silencing genes are known in the art. Forexample, expressed protein activity can be down-regulated or eliminatedusing antisense oligonucleotides, protein aptamers, nucleotide aptamers,and RNA interference (RNAi) (e.g., small interfering RNAs (siRNA), shorthairpin RNA (shRNA), and micro RNAs (miRNA) (see e.g., Fanning andSymonds (2006) Handb Exp Pharmacol. 173, 289-303G, describing hammerheadribozymes and small hairpin RNA; Helene, C., et al. (1992) Ann. N.Y.Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15, describingtargeting deoxyribonucleotide sequences; Lee et al. (2006) Curr OpinChem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) NatureBiotechnology 22(3), 326-330, describing RNAi; Pushparaj and Melendez(2006) Clinical and Experimental Pharmacology and Physiology 33(5-6),504-510, describing RNAi; Dillon et al. (2005) Annual Review ofPhysiology 67, 147-173, describing RNAi; Dykxhoorn and Lieberman (2005)Annual Review of Medicine 56, 401-423, describing RNAi). RNAi moleculesare commercially available from a variety of sources (e.g., Ambion, TX;Sigma Aldrich, MO; Invitrogen). Several siRNA molecule design programsusing a variety of algorithms are known to the art (see e.g., Cenixalgorithm, Ambion; BLOCK-iT™ RNAi Designer, Invitrogen; siRNA WhiteheadInstitute Design Tools, Bioinofrmatics & Research Computing). Traitsinfluential in defining optimal siRNA sequences include G/C content atthe termini of the siRNAs, Tm of specific internal domains of the siRNA,siRNA length, position of the target sequence within the CDS (codingregion), and nucleotide content of the 3′ overhangs.

Formulation

The agents and compositions described herein can be formulated by anyconventional manner using one or more pharmaceutically acceptablecarriers or excipients as described in, for example, Remington'sPharmaceutical Sciences (A. R. Gennaro, Ed.), 21st edition, ISBN:0781746736 (2005), incorporated herein by reference in its entirety.Such formulations will contain a therapeutically effective amount of abiologically active agent described herein, which can be in purifiedform, together with a suitable amount of carrier so as to provide theform for proper administration to the subject.

The term “therapeutically effective amount” can refers to an amount of acompound that, when administered to a subject for treating a disease, issufficient, in combination with another agent or alone, in one or moredoses, to effect such treatment for the disease. The “therapeuticallyeffective amount” can vary depending on the compound, the severity ofdisease, and the age, weight, etc., of the subject to be treated. Forthe purposes of the present disclosure, a “therapeutically effectiveamount of a JAK inhibitor” refers to an amount between about 0.01 mg/kgto about 100 mg/kg of body weight per day, preferably about 0.1 mg/kg toabout 10 mg/kg of body weight per day, or an equivalent dosageadministered more or less frequently.

In some embodiments, a therapeutically effective amount of a JAKinhibitor can be about 0.01 mg/kg to about 100 mg/kg of body weight perday. For example, the device diameter can be about 1 mg/kg about 2 mg/kgabout 3 mg/kg about 4 mg/kg about 5 mg/kg about 6 mg/kg about 7 mg/kgabout 8 mg/kg; about 9 mg/kg; about 10 mg/kg, about 11 mg/kg; about 12mg/kg, about 13 mg/kg, about 14 mg/kg; about 15 mg/kg; about 16 mg/kg;about 17 mg/kg; about 18 mg/kg; about 19 mg/kg; about 20 mg/kg; about 21mg/kg; about 22 mg/kg; about 23 mg/kg; about 24 mg/kg; about 25 mg/kg;about 26 mg/kg; about 27 mg/kg; about 28 mg/kg; about 29 mg/kg; about 30mg/kg; about 31 mg/kg; about 32 mg/kg; about 33 mg/kg; about 34 mg/kg;about 35 mg/kg; about 36 mg/kg; about 37 mg/kg; about 38 mg/kg; about 39mg/kg; about 40 mg/kg; about 41 mg/kg; about 42 mg/kg; about 43 mg/kg;about 44 mg/kg; about 45 mg/kg; about 46 mg/kg; about 47 mg/kg; about 48mg/kg; about 49 mg/kg; about 50 mg/kg; about 51 mg/kg; about 52 mg/kg;about 53 mg/kg; about 54 mg/kg; about 55 mg/kg; about 56 mg/kg; about 57mg/kg; about 58 mg/kg; about 59 mg/kg; about 60 mg/kg; about 61 mg/kg;about 62 mg/kg; about 63 mg/kg; about 64 mg/kg; about 65 mg/kg; about 66mg/kg; about 67 mg/kg; about 68 mg/kg; about 69 mg/kg; about 70 mg/kg;about 71 mg/kg; about 72 mg/kg; about 73 mg/kg; about 74 mg/kg; about 75mg/kg; about 76 mg/kg; about 77 mg/kg; about 78 mg/kg; about 79 mg/kg;about 80 mg/kg; about 81 mg/kg; about 82 mg/kg; about 83 mg/kg; about 84mg/kg; about 85 mg/kg; about 86 mg/kg; about 87 mg/kg; about 88 mg/kg;about 89 mg/kg; about 90 mg/kg; about 91 mg/kg; about 92 mg/kg; about 93mg/kg; about 94 mg/kg; about 95 mg/kg; about 96 mg/kg; about 97 mg/kg;about 98 mg/kg; about 99 mg/kg; about 100 mg/kg; about 101 mg/kg; about102 mg/kg; about 103 mg/kg; about 104 mg/kg; about 105 mg/kg; about 106mg/kg; about 107 mg/kg; about 108 mg/kg; about 109 mg/kg; about 110mg/kg; about 111 mg/kg; about 112 mg/kg; about 113 mg/kg; about 114mg/kg; about 115 mg/kg; about 116 mg/kg; about 117 mg/kg; about 118mg/kg; about 119 mg/kg; about 120 mg/kg; about 121 mg/kg; about 122mg/kg; about 123 mg/kg; about 124 mg/kg; about 125 mg/kg; about 126mg/kg; about 127 mg/kg; about 128 mg/kg; about 129 mg/kg; about 130mg/kg; about 131 mg/kg; about 132 mg/kg; about 133 mg/kg; about 134mg/kg; about 135 mg/kg; about 136 mg/kg; about 137 mg/kg; about 138mg/kg; about 139 mg/kg; about 140 mg/kg; about 141 mg/kg; about 142mg/kg; about 143 mg/kg; about 144 mg/kg; about 145 mg/kg; about 146mg/kg; about 147 mg/kg; about 148 mg/kg; about 149 mg/kg; about 150mg/kg; about 151 mg/kg; about 152 mg/kg; about 153 mg/kg; about 154mg/kg; about 155 mg/kg; about 156 mg/kg; about 157 mg/kg; about 158mg/kg; about 159 mg/kg; about 160 mg/kg; about 161 mg/kg; about 162mg/kg; about 163 mg/kg; about 164 mg/kg; about 165 mg/kg; about 166mg/kg; about 167 mg/kg; about 168 mg/kg; about 169 mg/kg; about 170mg/kg; about 171 mg/kg; about 172 mg/kg; about 173 mg/kg; about 174mg/kg; about 175 mg/kg; about 176 mg/kg; about 177 mg/kg; about 178mg/kg; about 179 mg/kg; about 180 mg/kg; about 181 mg/kg; about 182mg/kg; about 183 mg/kg; about 184 mg/kg; about 185 mg/kg; about 186mg/kg; about 187 mg/kg; about 188 mg/kg; about 189 mg/kg; about 190mg/kg; about 191 mg/kg; about 192 mg/kg; about 193 mg/kg; about 194mg/kg; about 195 mg/kg; about 196 mg/kg; about 197 mg/kg; about 198mg/kg; about 199 mg/kg; or about 200 mg/kg. Recitation of each of thesediscrete values is understood to include ranges between each value.

In some embodiments, therapeutically effective amount of a JAK inhibitormay be about 0.01 to about 100 mg/kg of body weight per day, preferablyabout 0.1 to about 10 mg/kg of body weight per day. In some embodiments,a therapeutically effective amount of a JAK inhibitor may be about 0.1to about 1 mg/kg of body weight per day. In other embodiments, atherapeutically effective amount of a JAK inhibitor may be about 0.25mg/kg to about 2.5 mg/kg of body weight per day. In yet otherembodiments, a therapeutically effective amount of a JAK inhibitor maybe about 0.5 mg/kg to about 5 mg/kg of body weight per day. In stillother embodiments, a therapeutically effective amount of a JAK inhibitormay be about 0.75 mg/kg to about 7.5 mg/kg of body weight per day. Indifferent embodiments, a therapeutically effective amount of a JAKinhibitor may be about 1 mg/kg to about 10 mg/kg of body weight per day.Recitation of each of these ranges is understood to include discretevalues between each value in the range.

Although the term “therapeutically effective amount” is described as anamount of a JAK inhibitor per day, a skilled artisan will appreciatethat a daily amount may be divided into one or more dosages to beadministered once, twice, three times or more daily. Alternatively, itmay be desirable to administer the JAK inhibitor on a less frequentbasis (e.g. once, twice, or three times per week; or once, twice, orthree times per month). A skilled artisan will appreciate that thefrequency of administration may influence the amount to be administered(e.g. generally speaking, monthly administration>weeklyadministration>daily administration>multiple times per day), and canformulate the pharmaceutical composition to provide a therapeuticallyeffective amount of the JAK inhibitor based on the disclosures herein.

Another aspect of the present disclosure provides administering one ormore “additional active ingredients” in combination with a JAKinhibitor. The additional active ingredient may be administered by thesame or different route of administration. When administered by the sameroute of administration, the additional active ingredient may beformulated with a JAK inhibitor (i.e., same pharmaceutical composition),or separately from the JAK inhibitor. Non-limiting examples ofadditional active ingredients that may be administered in combinationwith a JAK inhibitor of the present disclosure includes, but is notlimited to, antihistamines (e.g. cetirizine, diphenhydramine, doxepin,fexofenadine, hydroxyzine, loratadine, desloratidine), corticosteroids(e.g. triamcinolone, hydrocortisone, prednisone), local anesthetics(e.g. benzocaine, capsaicin, diperodon, lidocaine, menthol, polidocanol,pramoxine, prilocaine), topical immunomodulators (e.g. calcineurininhibitors, such as pimecrolimus, tacrolimus), μ-opioid receptorantagonists and K-opioid receptor agonists (e.g. butorphanol,nalfurafine, methylnaltrexone, nalmefene, naltrexone), antibiotics(rifampicin), cholestyramine, salicylic acid, antidepressants (e.g.fluvoxamine, mirtazapine, paroxetine, sertraline), and neuroleptics(e.g. gabapentin, pregablin).

The term “formulation” refers to preparing a drug in a form suitable foradministration to a subject, such as a human. Thus, a “formulation” caninclude pharmaceutically acceptable excipients, including diluents orcarriers.

The term “pharmaceutically acceptable” as used herein can describesubstances or components that do not cause unacceptable losses ofpharmacological activity or unacceptable adverse side effects. Examplesof pharmaceutically acceptable ingredients can be those havingmonographs in United States Pharmacopeia (USP 29) and National Formulary(NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md.,2005 (“USP/NF”), or a more recent edition, and the components listed inthe continuously updated Inactive Ingredient Search online database ofthe FDA. Other useful components that are not described in the USP/NF,etc. may also be used.

The term “pharmaceutically acceptable excipient,” as used herein, caninclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic, or absorption delaying agents. The useof such media and agents for pharmaceutical active substances is wellknown in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofaras any conventional media or agent is incompatible with an activeingredient, its use in the therapeutic compositions is contemplated.Supplementary active ingredients can also be incorporated into thecompositions.

A “stable” formulation or composition can refer to a composition havingsufficient stability to allow storage at a convenient temperature, suchas between about 0° C. and about 60° C., for a commercially reasonableperiod of time, such as at least about one day, at least about one week,at least about one month, at least about three months, at least aboutsix months, at least about one year, or at least about two years.

The formulation should suit the mode of administration. The agents ofuse with the current disclosure can be formulated by known methods foradministration to a subject using several routes which include, but arenot limited to, parenteral, pulmonary, oral, topical, intradermal,intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal,epidural, ophthalmic, buccal, and rectal. The individual agents may alsobe administered in combination with one or more additional agents ortogether with other biologically active or biologically inert agents.Such biologically active or inert agents may be in fluid or mechanicalcommunication with the agent(s) or attached to the agent(s) by ionic,covalent, Van der Waals, hydrophobic, hydrophilic or other physicalforces.

Controlled-release (or sustained-release) preparations may be formulatedto extend the activity of the agent(s) and reduce dosage frequency.Controlled-release preparations can also be used to effect the time ofonset of action or other characteristics, such as blood levels of theagent, and consequently affect the occurrence of side effects.Controlled-release preparations may be designed to initially release anamount of an agent(s) that produces the desired therapeutic effect, andgradually and continually release other amounts of the agent to maintainthe level of therapeutic effect over an extended period of time. Inorder to maintain a near-constant level of an agent in the body, theagent can be released from the dosage form at a rate that will replacethe amount of agent being metabolized or excreted from the body. Thecontrolled-release of an agent may be stimulated by various inducers,e.g., change in pH, change in temperature, enzymes, water, or otherphysiological conditions or molecules.

Agents or compositions described herein can also be used in combinationwith other therapeutic modalities, as described further below. Thus, inaddition to the therapies described herein, one may also provide to thesubject other therapies known to be efficacious for treatment of thedisease, disorder, or condition.

Therapeutic Methods

Also provided is a process of treating chronic idiopathic pruritus in asubject in need thereof. The process comprises administration of atherapeutically effective amount of a JAK inhibitor, so as to prevent,reduce, or relieve the symptoms of pruritus.

As described herein, it is shown that patients with recalcitrant chronicitch markedly improve when treated with JAK inhibitors.

Another aspect of the present disclosure provides treatment of chronicidiopathic pruritus includes. In some embodiments, a method of thepresent disclosure causes the clinical symptoms of CIP not to develop ina subject that may be predisposed to the disease (i.e., preventspruritus). In other embodiments, a method of the present disclosurearrests or reduces the progression of CIP or a symptom of CIP. In otherembodiments, a method of the present disclosure causes regression of CIPor symptom of CIP.

In certain embodiments, the number of Itch-Free Days in a subject isincreased at the end of the treatment period as compared to beginning ofthe treatment period. For example, the number of Itch-Free Days mayincrease by at least 1, at least 2, at least 3, at least 4, at least 5,at least 6, at least 7 days, at least 8 days, or at least 9 days.Alternatively, the number of Itch-Free Days may increase by at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16 days, at least 17 days, at least 18 days, or at least 19 days.

In certain embodiments, the severity of itching in a subject isdecreased at the end of the treatment period as compared to thebeginning of the treatment period. For example, a VAS score of itchingmay decrease by about 0.5, about 1.0, or about 1.5. Alternatively, a VASscore of itching may decrease by about 1.5, about 2.0, or about 2.5, orabout 3.0. In another alternative, a VAS score of itching may decreaseby about 3.0, about 3.5, about 4.0, about 4.5. In yet anotheralternative, itching severity may improve from extremely severe tosevere, preferably from extremely severe to moderate, or more preferablyfrom extremely severe to mild. In still another alternative, itchingseverity may improve from severe to moderate, or preferably from severeto mild. In another example, itching severity may improve from moderateto mild.

In certain embodiments, the quality of life of a subject is decreased atthe end of the treatment period as compared to the beginning of thetreatment period.

A skilled artisan will appreciate that the route of administration mayinfluence the amount that is needed to be administered in order toachieve a therapeutic effect. Generally speaking,oral>IV>transdermal/transmucosal>intranasal>intrathecal/epidural.

Those skilled in the art will appreciate that dosages may also bedetermined with guidance from Goodman & Goldman's The PharmacologicalBasis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493,and the Physicians' Desk Reference.

Methods described herein are generally performed on a subject in needthereof. A subject in need of the therapeutic methods described hereincan be a subject having, diagnosed with, suspected of having, or at riskfor developing pruritus. A determination of the need for treatment willtypically be assessed by a history and physical exam consistent with thedisease or condition at issue. Diagnosis of the various conditionstreatable by the methods described herein is within the skill of theart. The subject can be an animal subject, including a mammal, such ashorses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, hamsters,guinea pigs, and chickens, and humans. For example, the subject can be ahuman subject. As another example, a subject can be a mammal. A mammalcan include, but is not limited to a human, a companion animal, alivestock animal, a zoo animal, or a research animal. Non-limitingexamples of companion animals include a dog or a cat. Non-limitingexample of a livestock animal include a cow, a pig, a horse, a sheep ora goat. Non-limiting examples of a research animal include a non-humanprimate or a rodent.

The term “treating,” as used herein, can refer to controlling orpreventing the progression of chronic idiopathic pruritus. The term“controlling”, “treating” or “treatment” of chronic idiopathic prurituscan include: (1) preventing chronic idiopathic pruritus (i.e., causingthe clinical symptoms or signs of chronic idiopathic pruritus not todevelop in a subject that may be predisposed to the disease but does notyet experience or display symptoms/signs of the disease); (2) inhibitingthe chronic idiopathic pruritus (i.e., arresting or reducing theprogression of the disease or its clinical symptoms or signs); or (3)relieving the chronic idiopathic pruritus (i.e., causing regression ofthe disease or its clinical symptoms or signs).

Generally, a safe and effective amount of a JAK inhibitor is, forexample, that amount that would cause the desired therapeutic effect ina subject while minimizing undesired side effects. In variousembodiments, an effective amount of a JAK inhibitor described herein cansubstantially inhibit pruritus, slow the progress of pruritus, or limitthe development of pruritus.

According to the methods described herein, administration can beparenteral, pulmonary, oral, topical, intradermal, intramuscular,intraperitoneal, intravenous, intrathecally, subcutaneous, intranasal,epidural, ophthalmic, buccal, or rectal administration.

Parenteral administration, as used herein, can be administration byinjection, infusion or implantation. Non-limiting examples of includeepidural, intra-arterial, intracardiac, intramuscular, intraperitoneal,intraspinal, intrathoracic, intrathecal, intravenous, or subcutaneoustechniques.

When used in the treatments described herein, a therapeuticallyeffective amount of a JAK inhibitor can be employed in pure form or,where such forms exist, in pharmaceutically acceptable salt form andwith or without a pharmaceutically acceptable excipient. For example,the compounds of the present disclosure can be administered, at areasonable benefit/risk ratio applicable to any medical treatment, in asufficient amount to reduce, prevent, or treat pruritus.

The amount of a composition described herein that can be combined with apharmaceutically acceptable carrier to produce a single dosage form willvary depending upon the host treated and the particular mode ofadministration. It will be appreciated by those skilled in the art thatthe unit content of agent contained in an individual dose of each dosageform need not in itself constitute a therapeutically effective amount,as the necessary therapeutically effective amount could be reached byadministration of a number of individual doses.

Toxicity and therapeutic efficacy of compositions described herein canbe determined by standard pharmaceutical procedures in cell cultures orexperimental animals for determining the LD₅₀ (the dose lethal to 50% ofthe population) and the ED₅₀, (the dose therapeutically effective in 50%of the population). The dose ratio between toxic and therapeutic effectsis the therapeutic index that can be expressed as the ratio LD₅₀/ED₅₀,where larger therapeutic indices are generally understood in the art tobe optimal.

The specific therapeutically effective dose level for any particularsubject will depend upon a variety of factors including the disorderbeing treated and the severity of the disorder; activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the subject; the time ofadministration; the route of administration; the rate of excretion ofthe composition employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts (see e.g., Koda-Kimble etal. (2004) Applied Therapeutics: The Clinical Use of Drugs, LippincottWilliams & Wilkins, ISBN 0781748453; Winter (2003) Basic ClinicalPharmacokinetics, 4^(th) ed., Lippincott Williams & Wilkins, ISBN0781741475; Sharqel (2004) Applied Biopharmaceutics & Pharmacokinetics,McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is wellwithin the skill of the art to start doses of the composition at levelslower than those required to achieve the desired therapeutic effect andto gradually increase the dosage until the desired effect is achieved.If desired, the effective daily dose may be divided into multiple dosesfor purposes of administration. Consequently, single dose compositionsmay contain such amounts or submultiples thereof to make up the dailydose. It will be understood, however, that the total daily usage of thecompounds and compositions of the present disclosure will be decided byan attending physician within the scope of sound medical judgment.

Again, each of the states, diseases, disorders, and conditions,described herein, as well as others, can benefit from compositions andmethods described herein. Generally, treating a state, disease,disorder, or condition includes preventing or delaying the appearance ofclinical symptoms in a mammal that may be afflicted with or predisposedto the state, disease, disorder, or condition but does not yetexperience or display clinical or subclinical symptoms thereof. Treatingcan also include inhibiting the state, disease, disorder, or condition,e.g., arresting or reducing the development of the disease or at leastone clinical or subclinical symptom thereof. Furthermore, treating caninclude relieving the disease, e.g., causing regression of the state,disease, disorder, or condition or at least one of its clinical orsubclinical symptoms. A benefit to a subject to be treated can be eitherstatistically significant or at least perceptible to the subject or to aphysician.

Administration of a JAK inhibitor can occur as a single event or over atime course of treatment. For example, a JAK inhibitor can beadministered daily, weekly, bi-weekly, or monthly. For treatment ofacute conditions, the time course of treatment will usually be at leastseveral days. Certain conditions could extend treatment from severaldays to several weeks. For example, treatment could extend over oneweek, two weeks, or three weeks. For more chronic conditions, treatmentcould extend from several weeks to several months or even a year ormore.

Treatment in accord with the methods described herein can be performedprior to, concurrent with, or after conventional treatment modalitiesfor pruritus. Conventional treatments can include topical antipruriticsin the form of creams and sprays are often available over-the-counter ororal anti-itch drugs. For example, conventional treatments can includeantihistamines, such as diphenhydramine (Benadryl); corticosteroids(such as hydrocortisone topical cream, a topical steroid);counterirritants, such as mint oil, menthol, or camphor; crotamiton(trade name Eurax); local anesthetics, such as benzocaine topical cream(Lanacane); phototherapy (e.g., UVB), maintaining adequate skinmoisture; or topical emollients. Furthermore, TRPV1 inhibitors,dupilumab, or secukinumab can be used to treat itch.

A JAK inhibitor can be administered simultaneously or sequentially withanother agent, such as a, antibiotic, an antiinflammatory, or anotheragent (e.g., any of the above in the preceding paragraph). For example,a JAK inhibitor can be administered simultaneously with another agent,such as an antibiotic or an antiinflammatory. Simultaneousadministration can occur through administration of separatecompositions, each containing one or more of a JAK inhibitor, anantibiotic, an antiinflammatory, or another agent. Simultaneousadministration can occur through administration of one compositioncontaining two or more of a JAK inhibitor, an antibiotic, anantiinflammatory, or another agent. A JAK inhibitor can be administeredsequentially with an antibiotic, an antiinflammatory, or another agent.For example, a JAK inhibitor can be administered before or afteradministration of an antibiotic, an antiinflammatory, or another agent.

Administration

Another aspect of the present disclosure provides systemicallyadministering a therapeutically effective amount of a JAK inhibitor fora period of time.

Agents and compositions described herein can be administered accordingto methods described herein in a variety of means known to the art. Theagents and composition can be used therapeutically either as exogenousmaterials or as endogenous materials. Exogenous agents are thoseproduced or manufactured outside of the body and administered to thebody. Endogenous agents are those produced or manufactured inside thebody by some type of device (biologic or other) for delivery within orto other organs in the body.

Route of Administration

JAK inhibitor(s) may be formulated into pharmaceutical compositions andsystemically administered by a number of different means to deliver atherapeutically effective amount. Such compositions may be administered,for example, by nasal administration, oral administration, parenteraladministration, rectal administration, topical administration,transdermal administration, or transmucosal administration, in dosageunit formulations containing conventional nontoxic pharmaceuticallyacceptable carriers, adjuvants, and vehicles as desired. Formulation ofdrugs is discussed in, for example, Hoover, John E., Remington'sPharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (1975), andLiberman, H. A. and Lachman. L., Eds., Pharmaceutical Dosage Forms,Marcel Decker, New York, N.Y. (1980).

Preparations for oral administration generally contain inert excipientsin addition to the active pharmaceutical ingredient. Oral preparationsmay be enclosed in gelatin capsules or compressed into tablets. Commonexcipients used in such preparations include pharmaceutically compatiblefillers/diluents such as microcrystalline cellulose, hydroxypropylmethylcellulose, starch, lactose, sucrose, glucose, mannitol, sorbitol,dibasic calcium phosphate, or calcium carbonate; binding agents such asalginic acid, carboxymethylcellulose, microcrystalline cellulose,gelatin, gum tragacanth, or polyvinylpyrrolidone; disintegrating agentssuch as alginic acid, cellulose, starch, or polyvinylpyrrolidone;lubricants such as calcium stearate, magnesium stearate, talc, silica,or sodium stearyl fumarate; glidants such as colloidal silicon dioxide;sweetening agents such as sucrose or saccharin; flavoring agents such aspeppermint, methyl salicylate, or citrus flavoring; coloring agents; andpreservatives such as antioxidants (e.g., vitamin A, vitamin C, vitaminE, or retinyl palmitate), citric acid, or sodium citrate. Oralpreparations may also be administered as aqueous suspensions, elixirs,or syrups. For these, the active ingredient may be combined with varioussweetening or flavoring agents, coloring agents, and, if so desired,emulsifying and/or suspending agents, as well as diluents such as water,ethanol, glycerin, and combinations thereof.

For parenteral administration, the preparation may be an aqueous or anoil-based solution. Aqueous solutions may include a sterile diluent suchas water, saline solution, a pharmaceutically acceptable polyol such asglycerol, propylene glycol, or other synthetic solvents; anantibacterial and/or antifungal agent such as benzyl alcohol, methylparaben, chlorobutanol, phenol, thimerosal, and the like; an antioxidantsuch as ascorbic acid or sodium bisulfite; a chelating agent such asetheylenediaminetetraacetic acid; a buffer such as acetate, citrate, orphosphate; and/or an agent for the adjustment of tonicity such as sodiumchloride, dextrose, or a polyalcohol such as mannitol or sorbitol. ThepH of the aqueous solution may be adjusted with acids or bases such ashydrochloric acid or sodium hydroxide. Oil-based solutions orsuspensions may further comprise sesame, peanut, olive oil, or mineraloil. The composition may also be provided in a powder form forreconstitution with a suitable vehicle including, but not limited to,sterile, pyrogen-free water.

For topical, transdermal or transmucosal administration, penetrantsappropriate to the barrier to be permeated are generally included in thepreparation. Transmucosal administration may be accomplished through theuse of nasal sprays, aerosol sprays, tablets, or suppositories, andtransdermal administration may be via ointments, salves, gels, patches,or creams as generally known in the art. Non-limiting examples ofsuitable carriers for transdermal embodiments include mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax, sorbitan monostearate,Polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol,benzyl alcohol, and water. For these embodiments, the molecular weightof the composition may range from about 1 to about 50 Daltons

For nasal administration, compositions may be formulated as a solution,suspension, or emulsion that may be administered as a dry powder or inthe form of an aerosol using a propellant, such asdichlorodifluoromethane or trichlorofluoromethane. A nasal spray caninclude a saline spray.

In some embodiments, the route of administration is chosen fromsubcutaneous, epidural, intrathecal, intravenous, nasal, oral, ortopical. In other preferred embodiments, the route of administration ischosen from intrathecal and nasal.

As discussed above, administration can be parenteral, pulmonary, oral,topical, intradermal, intramuscular, intraperitoneal, intravenous,subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectaladministration.

Delivery Methods

Agents and compositions described herein can be administered in avariety of methods well known in the arts. Administration can include,for example, methods involving oral ingestion, direct injection (e.g.,systemic or stereotactic), implantation of cells engineered to secretethe factor of interest, drug-releasing biomaterials, polymer matrices,gels, permeable membranes, osmotic systems, multilayer coatings,microparticles, implantable matrix devices, mini-osmotic pumps,implantable pumps, injectable gels and hydrogels, liposomes, micelles(e.g., up to 30 μm), nanospheres (e.g., less than 1 μm), microspheres(e.g., 1-100 μm), reservoir devices, a combination of any of the above,or other suitable delivery vehicles to provide the desired releaseprofile in varying proportions. Other methods of controlled-releasedelivery of agents or compositions will be known to the skilled artisanand are within the scope of the present disclosure.

Delivery systems may include, for example, an infusion pump which may beused to administer the agent or composition in a manner similar to thatused for delivering insulin or chemotherapy to specific organs ortumors. Typically, using such a system, an agent or composition can beadministered in combination with a biodegradable, biocompatiblepolymeric implant that releases the agent over a controlled period oftime at a selected site. Examples of polymeric materials includepolyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid,polyethylene vinyl acetate, and copolymers and combinations thereof. Inaddition, a controlled release system can be placed in proximity of atherapeutic target, thus requiring only a fraction of a systemic dosage.

Agents can be encapsulated and administered in a variety of carrierdelivery systems. Examples of carrier delivery systems includemicrospheres, hydrogels, polymeric implants, smart polymeric carriers,and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006)Polymers in Drug Delivery, CRC, ISBN-10: 0849325331). Carrier-basedsystems for molecular or biomolecular agent delivery can: provide forintracellular delivery; tailor biomolecule/agent release rates; increasethe proportion of biomolecule that reaches its site of action; improvethe transport of the drug to its site of action; allow colocalizeddeposition with other agents or excipients; improve the stability of theagent in vivo; prolong the residence time of the agent at its site ofaction by reducing clearance; decrease the nonspecific delivery of theagent to nontarget tissues; decrease irritation caused by the agent;decrease toxicity due to high initial doses of the agent; alter theimmunogenicity of the agent; decrease dosage frequency, improve taste ofthe product; or improve shelf life of the product.

Treatment Period

According to the method of the invention, a therapeutically effectiveamount of a JAK inhibitor is administered to a subject in need thereoffor a period of time (“the treatment period”), sufficient to treat CIP.The period of time the JAK inhibitor is administered may be referred toas a “treatment period.”

In some embodiments, the amount of a JAK inhibitor and frequency ofadministration does not vary during a treatment period. For example, aJAK inhibitor may be administered daily for about 1, 2, 3, 4, 5, 6, or 7days. Alternatively, a JAK inhibitor may be administered daily for about2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In another alternative, aJAK inhibitor may be administered daily for 3 months, 6 months, 12months or more. In still other alternative, a JAK inhibitor may beadministered weekly for about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12weeks. In yet another alternative, a JAK inhibitor may be administeredweekly for 3 months, 6 months, 12 months or more. In anotheralternative, a JAK inhibitor may be administered monthly for 3 months, 6months, 12 months or more. Alternatively, a JAK inhibitor may beadministered on an “as needed” basis.

In other embodiments, the amount a JAK inhibitor and frequency ofadministration can vary during a treatment period. For example, atreatment period may comprise a first phase and a second phase, wherein(a) an amount of a JAK inhibitor administered in the first phase isgreater than an amount of the JAK inhibitor administered in the secondphase; (b) a JAK inhibitor is administered more frequently in the firstis greater in the second phase; or (c) an amount of a JAK inhibitoradministered in the first phase is greater than an amount of the JAKinhibitor administered in the second phase, and administration is morefrequent in the first phase than in the second phase. It is alsocontemplated that the route of administration differs between the firstand second phase. The period of time for the first phase may be about 2,3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, and the period of time for thesecond phase may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 weeks. Atreatment phase may also comprise more than two phases (e.g. 3, 4, 5, ormore phases).

Screening

Also provided are methods for screening for JAK inhibitors.

The subject methods find use in the screening of a variety of differentcandidate molecules (e.g., potentially therapeutic candidate molecules).Candidate substances for screening according to the methods describedherein include, but are not limited to, fractions of tissues or cells,nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers,ribozymes, triple helix compounds, antibodies, and small (e.g., lessthan about 2000 mw, or less than about 1000 mw, or less than about 800mw) organic molecules or inorganic molecules including but not limitedto salts or metals.

Candidate molecules encompass numerous chemical classes, for example,organic molecules, such as small organic compounds having a molecularweight of more than 50 and less than about 2,500 Daltons. Candidatemolecules can comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and typicallyinclude at least an amine, carbonyl, hydroxyl or carboxyl group, andusually at least two of the functional chemical groups. The candidatemolecules can comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups.

A candidate molecule can be a compound in a library database ofcompounds. One of skill in the art will be generally familiar with, forexample, numerous databases for commercially available compounds forscreening (see e.g., ZINC database, UCSF, with 2.7 million compoundsover 12 distinct subsets of molecules; Irwin and Shoichet (2005) J ChemInf Model 45, 177-182). One of skill in the art will also be familiarwith a variety of search engines to identify commercial sources ordesirable compounds and classes of compounds for further testing (seee.g., ZINC database; eMolecules.com; and electronic libraries ofcommercial compounds provided by vendors, for example: ChemBridge,Princeton BioMolecular, Ambinter SARL, Enamine, ASDI, Life Chemicalsetc.).

Candidate molecules for screening according to the methods describedherein include both lead-like compounds and drug-like compounds. Alead-like compound is generally understood to have a relatively smallerscaffold-like structure (e.g., molecular weight of about 150 to about350 kD) with relatively fewer features (e.g., less than about 3 hydrogendonors and/or less than about 6 hydrogen acceptors; hydrophobicitycharacter x log P of about −2 to about 4) (see e.g., Angewante (1999)Chemie Int. ed. Engl. 24, 3943-3948). In contrast, a drug-like compoundis generally understood to have a relatively larger scaffold (e.g.,molecular weight of about 150 to about 500 kD) with relatively morenumerous features (e.g., less than about 10 hydrogen acceptors and/orless than about 8 rotatable bonds; hydrophobicity character x log P ofless than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44,235-249). Initial screening can be performed with lead-like compounds.

When designing a lead from spatial orientation data, it can be useful tounderstand that certain molecular structures are characterized as being“drug-like”. Such characterization can be based on a set of empiricallyrecognized qualities derived by comparing similarities across thebreadth of known drugs within the pharmacopoeia. While it is notrequired for drugs to meet all, or even any, of these characterizations,it is far more likely for a drug candidate to meet with clinicalsuccessful if it is drug-like.

Several of these “drug-like” characteristics have been summarized intothe four rules of Lipinski (generally known as the “rules of fives”because of the prevalence of the number 5 among them). While these rulesgenerally relate to oral absorption and are used to predictbioavailability of compound during lead optimization, they can serve aseffective guidelines for constructing a lead molecule during rationaldrug design efforts such as may be accomplished by using the methods ofthe present disclosure.

The four “rules of five” state that a candidate drug-like compoundshould have at least three of the following characteristics: (i) aweight less than 500 Daltons; (ii) a log of P less than 5; (iii) no morethan 5 hydrogen bond donors (expressed as the sum of OH and NH groups);and (iv) no more than 10 hydrogen bond acceptors (the sum of N and Oatoms). Also, drug-like molecules typically have a span (breadth) ofbetween about 8 Å to about 15 Å.

Kits

Also provided are kits. Such kits can include an agent or compositiondescribed herein and, in certain embodiments, instructions foradministration. Such kits can facilitate performance of the methodsdescribed herein. When supplied as a kit, the different components ofthe composition can be packaged in separate containers and admixedimmediately before use. Components include, but are not limited to JAKinhibitors and components for delivery of the JAK inhibitor. Suchpackaging of the components separately can, if desired, be presented ina pack or dispenser device which may contain one or more unit dosageforms containing the composition. The pack may, for example, comprisemetal or plastic foil such as a blister pack. Such packaging of thecomponents separately can also, in certain instances, permit long-termstorage without losing activity of the components.

Kits may also include reagents in separate containers such as, forexample, sterile water or saline to be added to a lyophilized activecomponent packaged separately. For example, sealed glass ampules maycontain a lyophilized component and in a separate ampule, sterile water,sterile saline or sterile each of which has been packaged under aneutral non-reacting gas, such as nitrogen. Ampules may consist of anysuitable material, such as glass, organic polymers, such aspolycarbonate, polystyrene, ceramic, metal or any other materialtypically employed to hold reagents. Other examples of suitablecontainers include bottles that may be fabricated from similarsubstances as ampules, and envelopes that may consist of foil-linedinteriors, such as aluminum or an alloy. Other containers include testtubes, vials, flasks, bottles, syringes, and the like. Containers mayhave a sterile access port, such as a bottle having a stopper that canbe pierced by a hypodermic injection needle. Other containers may havetwo compartments that are separated by a readily removable membrane thatupon removal permits the components to mix. Removable membranes may beglass, plastic, rubber, and the like.

In certain embodiments, kits can be supplied with instructionalmaterials. Instructions may be printed on paper or other substrate,and/or may be supplied as an electronic-readable medium, such as afloppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, and the like. Detailed instructions may not be physicallyassociated with the kit; instead, a user may be directed to an Internetweb site specified by the manufacturer or distributor of the kit.

Compositions and methods described herein utilizing molecular biologyprotocols can be according to a variety of standard techniques known tothe art (see, e.g., Sambrook and Russel (2006) Condensed Protocols fromMolecular Cloning: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols inMolecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929;Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3ded., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J.and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005)Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production ofRecombinant Proteins: Novel Microbial and Eukaryotic Expression Systems,Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein ExpressionTechnologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions and methods described herein are provided to better definethe present disclosure and to guide those of ordinary skill in the artin the practice of the present disclosure. Unless otherwise noted, termsare to be understood according to conventional usage by those ofordinary skill in the relevant art.

In some embodiments, numbers expressing quantities of ingredients,properties such as molecular weight, reaction conditions, and so forth,used to describe and claim certain embodiments of the present disclosureare to be understood as being modified in some instances by the term“about.” In some embodiments, the term “about” is used to indicate thata value includes the standard deviation of the mean for the device ormethod being employed to determine the value. In some embodiments, thenumerical parameters set forth in the written description and attachedclaims are approximations that can vary depending upon the desiredproperties sought to be obtained by a particular embodiment. In someembodiments, the numerical parameters should be construed in light ofthe number of reported significant digits and by applying ordinaryrounding techniques. Notwithstanding that the numerical ranges andparameters setting forth the broad scope of some embodiments of thepresent disclosure are approximations, the numerical values set forth inthe specific examples are reported as precisely as practicable. Thenumerical values presented in some embodiments of the present disclosuremay contain certain errors necessarily resulting from the standarddeviation found in their respective testing measurements. The recitationof ranges of values herein is merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range. Unless otherwise indicated herein, each individual value isincorporated into the specification as if it were individually recitedherein.

In some embodiments, the terms “a” and “an” and “the” and similarreferences used in the context of describing a particular embodiment(especially in the context of certain of the following claims) can beconstrued to cover both the singular and the plural, unless specificallynoted otherwise. In some embodiments, the term “or” as used herein,including the claims, is used to mean “and/or” unless explicitlyindicated to refer to alternatives only or the alternatives are mutuallyexclusive.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and can also cover other unlisted steps. Similarly, anycomposition or device that “comprises,” “has” or “includes” one or morefeatures is not limited to possessing only those one or more featuresand can cover other unlisted features.

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.“such as”) provided with respect to certain embodiments herein isintended merely to better illuminate the present disclosure and does notpose a limitation on the scope of the present disclosure otherwiseclaimed. No language in the specification should be construed asindicating any non-claimed element essential to the practice of thepresent disclosure.

Groupings of alternative elements or embodiments of the presentdisclosure disclosed herein are not to be construed as limitations. Eachgroup member can be referred to and claimed individually or in anycombination with other members of the group or other elements foundherein. One or more members of a group can be included in, or deletedfrom, a group for reasons of convenience or patentability. When any suchinclusion or deletion occurs, the specification is herein deemed tocontain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims.

Citation of a reference herein shall not be construed as an admissionthat such is prior art to the present disclosure.

Having described the present disclosure in detail, it will be apparentthat modifications, variations, and equivalent embodiments are possiblewithout departing the scope of the present disclosure defined in theappended claims. Furthermore, it should be appreciated that all examplesin the present disclosure are provided as non-limiting examples.

EXAMPLES

The following non-limiting examples are provided to further illustratethe present disclosure. It should be appreciated by those of skill inthe art that the techniques disclosed in the examples that followrepresent approaches the inventors have found function well in thepractice of the present disclosure, and thus can be considered toconstitute examples of modes for its practice. However, those of skillin the art should, in light of the present disclosure, appreciate thatmany changes can be made in the specific embodiments that are disclosedand still obtain a like or similar result without departing from thespirit and scope of the present disclosure.

Example 1: Immune Dysregulation Underlies a Subset of Patients withChronic Idiopathic Pruritus

Chronic pruritus is a highly debilitating condition thatdisproportionately affects the elderly, many of whom experience chronicidiopathic pruritus (CIP), or itch of unknown origin lasting greaterthan six weeks. Multiple factors are believed to underlie CIP, includingaging-associated skin barrier dysfunction, sensory neuropathy, andimmunosenescence, the waning of immune function that results in an“allergic,” T helper type 2 (Th2) cell response. However, theimmunologic profile of CIP patients remains poorly understood.

This example describes four elderly CIP patients with evidence of markedimmune dysregulation after exclusion of other cutaneous and systemiccauses of pruritus. Despite the lack of primary dermatologic processes,three out of four patients showed lymphocytic infiltrates on skinbiopsy, two of whom demonstrated increased eosinophils (see FIG. 2B andFIG. 2C, and TABLE 2). Immunoglobulin panels and blood flow cytometryrevealed previously unrecognized humoral and cellular immune defects,including T and B cell lymphopenia, eosinophilia, andhypogammaglobulinemia (see TABLE 2).

Given the patients' broad immunologic defects, underlying primaryimmunodeficiencies, like common variable immunodeficiency (CVID), wereinitially considered. However, the patients' reduced IgG values were notassociated with decreases in IgA or IgM, as in CVID. The patients alsolacked any history of CVID-associated complications, such as recurrentinfections, autoimmunity, and malignancy. These findings suggest thatCIP patients may acquire secondary immune defects resembling CVID as aconsequence of the aging process.

Immunosenescence results in a loss of T helper type 1 (Th1) cell-drivenprotective immunity that contributes to skewing towards an allergic, Th2response. Indeed, the CIP patients exhibited Th2 polarization asevidenced by their elevated serum IgE levels, peripheral eosinophilia,and/or tissue eosinophil infiltration (see TABLE 2). Additionally, theirCD8 lymphopenia confirms the loss of Th1 responses, further supportingage-dependent Th2 polarization as a potential contributor to thedevelopment of chronic itch. Strikingly, comparison to age-matchedcontrol populations indicates that these CIP patients suffer fromimmunologic imbalances in excess of similarly-aged peers. Three of thefour patients' CD8+ T cell counts were significantly reduced incomparison to control subjects of corresponding age; for example, theCD8+ T cell count of 67/mm3 in Case 3 was nearly two standard deviationsbelow the mean reported by Provinciali et al. Likewise, IgG levels fortwo of the four patients (Cases 1 and 3) fell below the bottom 2.5%described for age-matched controls. These findings suggest that CIPpatients may have a greater degree of immune dysfunction that results inan increased susceptibility to chronic pruritus, even above theincreased risk already recognized in the elderly.

In conclusion, these CIP patients exhibit immunologic deficits inassociation with Th2 polarization. These findings suggestaging-associated immunosenescence may promote immune dysregulation thatmay, in part, explain the increased prevalence of chronic idiopathicpruritus in the elderly. Thus, the restoration of immune homeostasis byrecombinant cytokine or immunoglobulin repletion may represent noveltherapeutic approaches for CIP.

TABLE 2 Legend: (a) Average and peak Numerical Rating Scale (NRS) itchscores at the time of presentation to our clinic are noted,respectively. (b) A thorough workup was performed to establish thediagnosis of CIP by exclusion of other cutaneous and systemic causes ofpruritus, including psychiatric and neurologic conditions, renalfailure, biliary dysfunction, thyroid abnormalities, HIV/AIDS, hepatitisB, and hepatitis C. None of the patients had a history of recurrentinfections, autoimmune conditions, or malignancy. (c) Notably, a courseof oral prednisone prescribed for Case 2's polymyalgia rheumaticaimproved his muscle weakness but did not resolve his pruritus. (d)Outside of two episodes of septic arthritis arising from kneereplacement surgery, the patient had no history of persistent orrecurrent infections, autoimmunity, or malignancy. (e) The patient inCase 3 had an initial IgE level of 329 IU/mL, but it normalized to 195IU/mL two months later.

TABLE 2 Summary of Clinical and Immunologic Characteristics. ReferenceCase 1 Case 2 Case 3 Case 4 Age —  75  84 77   90 Gender — M M M M NRSScore^(a) — 6, 10 10, 10 8, 10 8, 8 Duration of —  3    0.5 5  4Symptoms (years) Past Medical — — Polymyalgia Osteoarthritis —History^(b) Rheumatics Prior Treatments — — Topical Topical —emollients, emollients steroids^(c), oral steroids, oral gabaperatinantihistamines, doxepin, zolpidem History of — No No No^(d) No RecurrentInfection, Autoimmunity, or Malignancy? CBC % Lymphocytes   20-54.3% 18%↓ 8.3% ↓ 6.8% ↓ 18.2% ↓ Abs Eosinophil  0-0.5    1.8 Normal  1.3    1.8Count (K/mm³) Diagnostic Testing SPEP — Normal — Normal — CXR — — —Normal Nonspecific calcifications CT Scan — Normal — — — Bone Marrow —Normal — — Normal Biopsy Skin Biopsy — Sparse Dermal edema SuperficialSpongiosis superficial with minimal perivascular with lichenoidperivascular inflammation lymphocytic infiltrates and lymphocyticinflammation eosinophils infilterate with abundant eosinophilsImmunoglobulin Panel IgM (mg/dL) 30-210   61.5 122 52.1     <19.0 ↓ IgG(mg/dL) 700-1450   512 ↓   619 ↓ 552 ↓ 750 IgA (mg/dL) 70-370 212 16785.7    525 ↑ IgE (IU/mL)  0-250   448 ↑  1940 ↑ 329^(e) ↑   24.7 FlowCytometry Panel Abs CD8⁺ Count 211-977  359   60 ↓  67 ↓   96 ↓ (#/mm³)Abs CD4⁺ Count 393-1607 718   360 ↓ 233 ↓ 654 (#/mm³) Abs CD19⁺ Count70-545   87 ↓ 240  4 ↓   48 ↓ (#/mm³) Abs CD3⁺ Count 706-2482 1076   420 ↓ 300 ↓ 760 (#/mm³) ^(a)Average and peak Numerical Rating Scale(NRS) itch scores at the time of presentation to our clinic are noted,respectively. ^(b)A thorough workup was performed to establish thediagnosis of CIP by exclusion of other cutaneous and systemic causes ofpruritus, including psychiatric and neurologic conditions, renalfailure, biliary dysfunction, thyroid abnormalities, HIV/AIDS, hepatitisB, and hepatitis C. None of the patients had a history of recurrentinfections, autoimmune conditions, or malignancy. ^(c)Notably, a courseof oral prednisone prescribed for Case 2's polymyalgia rheumaticaimproved his muscle weakness but did not resolve his pruritus.^(d)Outside of two episodes of septic arthritis arising from kneereplacement surgery, the patient had no history of persistent ofrecurrent infections, autoimmunity, or malignancy. ^(e)The patient inCase 3 had a initial IgE level of 329 IU/mL, but it normalized to 195IU/mL two months later.

Example 2: Sensory Neurons Respond to Type 2 Cytokines AlongItch-Sensory Pathways

The proinflammatory type 2 cytokines IL-4, IL-5, and IL-13 are known topromote atopic dermatitis (AD) pathogenesis (Gittler et al., 2012;Weidinger and Novak, 2016). Importantly, Phase II and III clinicaltrials employing dupilumab, an IL-4/13 receptor (IL-4Rα) antagonist, tosuppress allergic inflammation in AD patients demonstrated remarkableimprovement in both disease and itch severity (Beck et al., 2014;Simpson et al., 2016; Thaçi et al., 2016). Although IL-4/13-dependentinflammatory processes are well-established mediators of skininflammation in AD, it is not known whether these cytokines directlypromote chronic itch. To examine whether type 2 cytokines can directlyactivate sensory neurons, we performed RT-PCR of dorsal root ganglia(DRG) from wild type (WT) mice and found expression of IL-4Rα (Il4ra)and IL-13 receptor alpha 1 (IL-13Rα1; Il13ra1), but not IL-5 receptoralpha (IL-5Rα; Il5ra) (see e.g., FIG. 5A). As expected, we were alsoable to detect expression of the receptor for the known cytokinepruritogen IL-31 (Il31ra) (see e.g., FIG. 5A) (Cevikbas et al., 2014;Dillon et al., 2004; Sonkoly et al., 2006). Similarly, DRG from humancadaveric donors also expressed IL-4Rα (IL4RA), IL-13Rα1 (IL13RA1), andIL-31RA (IL31RA), but not IL-5Rα (IL5RA) (see e.g., FIG. 5B).Strikingly, we found that sensory ganglia express much higher levels ofIl4ra compared to Il13ra1 and Il31ra (see e.g., FIG. 5C). Takentogether, these findings provoke the hypothesis that sensory neurons canbe directly stimulated by the type 2 cytokines IL-4 and IL-13.

To test whether sensory neurons can be activated by type 2 cytokines, weemployed ratiometric calcium imaging of murine DRG neurons. Ex vivostimulation of cultured neurons with recombinant murine IL-4 (see e.g.,FIG. 5D) or IL-13 (see e.g., FIG. 5E) demonstrated a rapid rise ofintracellular calcium in a subset of neurons. These responses weresimilar to those observed with IL-31 stimulation (see e.g., FIG. 5F). Wenext sought to comprehensively examine the composition of IL-4- andIL-13-responsive neurons in the DRG. Stimulation of cultured neuronswith IL-4 or IL-13 led to calcium responses in 4.1±0.5% and 3.3±0.8% oftotal DRG neurons, respectively (see e.g., FIG. 5G). Consistent withprevious reports (Cevikbas et al., 2014; Liu et al., 2009),approximately 5% and 25% of neurons were responsive to IL-31 andhistamine, respectively (see e.g., FIG. 5G). As expected given the lackof expression of Il5ra in the DRG (see e.g., FIG. 5A-B), no responseswere seen when DRG neurons were stimulated with IL-5 (see e.g., FIG.12). Collectively, these studies demonstrate that sensory neuronsrespond selectively to the type 2 cytokines IL-4 and IL-13.

We next performed calcium imaging of DRG neurons in response tosuccessive challenges with defined pruritogens to examine whether IL-4or IL-13 activate previously defined itch-sensory pathways. When DRGneurons were serially stimulated with IL-4, as well as with IL-31 andhistamine, 88.9% of IL-4-responsive neurons also responded to IL-31 orhistamine (see e.g., FIG. 5H). Similarly, 75.0% of IL-13-responsiveneurons also responded to IL-31 or histamine (see e.g., FIG. 5I).Together, these studies indicate that both IL-4 and IL-13 can activatefamilies of neurons that include previously defined itch-sensorypathways. However, we note that both IL-4 and IL-13 can also activatesensory neurons that do not respond to either IL-31 or histamine.

To validate our findings at the single-cell level, we reanalyzed apreviously published single-cell RNA-sequencing data set that correlatedthe transcriptional profile of single neurons to predicted sensorymodalities in mice (Usoskin et al., 2015). Reexamination of this dataset revealed that the families of small diameter neurons predicted tomediate itch (NP1, NP2, and NP3) are enriched in neurons that expressIl4ra and Il13ra1 compared to other families of small diameter neuronspredicted to mediate nociception (PEP1 and PEP2) and mechanoreception(TH) (see e.g., FIG. 13). Although previously defined pruriceptors suchas Mas-related G protein-coupled receptor A3 (MrgprA3) and IL-31RA arehighly specific to defined itch clusters such as NP2 and NP3,respectively (Usoskin et al., 2015), the expression of Il4ra spansmultiple pruriceptive clusters (see e.g., FIG. 13). These analyses andour functional calcium imaging findings indicate that type 2 cytokinescan activate multiple itch-sensory pathways simultaneously.Collectively, these studies provoke the hypothesis that type 2 cytokinespromote chronic itch via direct stimulation of neurons in addition totheir well-defined effects on immune cells.

Example 3: Type 2 Cytokine Receptor Expression is Enhanced in SensoryGanglia in the Setting of Chronic Itch

To explore the contribution of type 2 cytokines to itch in vivo, weemployed a previously established murine model of atopic dermatitis(AD)-like skin inflammation in which mice are treated with the topicalirritant calcipotriol (MC903) (see e.g., FIG. 6A) (Kim et al., 2013a,2014, Li et al., 2006, 2009; Morita et al., 2015; Noti et al., 2013,2014; Siracusa et al., 2011). Compared to control mice treated withvehicle only (ethanol, EtOH), MC903-treated mice develop robust skininflammation as measured by ear skin thickening (see e.g., FIG. 6B) andhistologic features of inflammation including stratum corneum thickening(hyperkeratosis), epidermal hyperplasia (acanthosis), and a mixed dermalinflammatory infiltrate (see e.g., FIG. 6C). Accordingly, using apreviously established histopathology grading system (Kim et al., 2014),we found that MC903-treated mice exhibit a higher histologic score ofinflammation (see e.g., FIG. 6D). Importantly, in addition to featuresof skin inflammation, MC903-treated mice demonstrate a marked chronicitch phenotype as measured by quantifying scratching bouts over time(see e.g., FIG. 6E).

To comprehensively examine the inflammatory changes induced in thesetting of AD-associated chronic itch, we performed RNA-sequencing ofskin samples from both control and MC903-treated mice. As expected, skinfrom MC903-treated mice demonstrated a distinct transcriptional programcompared to control skin including upregulation of key AD-associatedcytokine signaling molecule transcripts such as IL-4 (Il4), IL-13(Il13), IL-4Rα (Il4ra), and IL-13Rα1 (Il13ra1) (see e.g., FIG. 6F, seee.g., FIG. 15). Further, expression of Il4ra and Il13ra1, but not Il5ra,were also significantly upregulated in sensory ganglia from mice withAD-associated itch (see e.g., FIG. 6G). Thus, these data suggest thatthe sensory nervous system can selectively enhance type 2 cytokinesignaling via upregulation of receptors for both IL-4 and IL-13 in thesetting of chronic itch and provoke the hypothesis that dysregulatedtype 2 cytokine signaling in sensory neurons promotes pathologic chronicitch.

Example 4: Type 2 Immune Cells Directly Interact with Sensory NerveFibers

To examine whether type 2 cytokine-expressing cells come into closeproximity with sensory nerve fibers innervating the skin, we generatednovel dual reporter mice in which sensory nerve fibers express tdTomatounder a sensory neuron-specific (Na_(v)1.8) Cre recombinase (Agarwal etal., 2004) and type 2 immune cells express enhanced GFP (eGFP) under anIL-4 reporter (Mohrs et al., 2001). Therefore, these dual reporter miceallowed us to visualize whether type 2 immune cells (IL-4-eGFP⁺)interact with sensory nerve fibers (Na_(v)1.8-tdTomato⁺). We thentreated these dual reporter mice with vehicle control (EtOH) on one earand MC903 on the second ear and performed live intravital two-photonimaging of the skin of both ears (see e.g., FIG. 7A) (Zinselmeyer etal., 2009). Although we observed very few IL-4-eGFP⁺ cells in thecontrol skin (see e.g., FIG. 7B, FIG. 16), we found many motile type 2immune cells in close proximity to sensory nerve fibers in MC903-treatedskin (see e.g., FIG. 7C, FIG. 17). At the single cell level, we observedthat a subset of type 2 immune cells markedly reduced theirinstantaneous speed upon interacting with a sensory nerve fiber butresumed rapid transit after leaving the fiber (see e.g., FIG. 7D, FIG.18). Overall, we found that IL-4-eGFP⁺ type 2 immune cells associatedwith Na_(v)1.8-tdTomato⁺ sensory nerve fibers had a lower mean speedover the course of imaging compared to those that were not inassociation with sensory fibers (see e.g., FIG. 7E). Taken together,these findings suggest the existence of robust bidirectionalinteractions between the immune and sensory nervous systems that resultin both altered immune cell trafficking and activation of sensoryfibers. The observation that type 2 immune cells physically interactwith sensory fibers in vivo lends further support to the hypothesis thattype 2 cytokine signaling may mediate chronic itch via direct neuronalstimulation.

Example 5: Neuronal Type 2 Cytokine Signaling is Necessary for theDevelopment of Chronic Itch

To investigate the in vivo functions of sensory neuron-intrinsic type 2cytokine signaling, we generated mice that lack IL-4Rα specifically onsensory neurons using the Na_(v)1.8-Cre mice (Na_(v)1.8-Cre⁺Il4ra^(fl/fl), IL-4Rα^(Δneuron)) (see e.g., FIG. 8A). IL-4Rα^(Δneuron)mice were born at normal Mendelian frequencies, and deletion of Il4rawas confirmed in sensory ganglia (see e.g., FIG. 8B). We then treatedIL-4Rα^(Δneuron) mice with MC903 to explore the direct neuronalcontributions of IL-4/13 signaling to chronic itch (see e.g., FIG. 8A).Strikingly, upon induction of skin inflammation, IL-4Rα^(Δneuron) micedemonstrated a significant reduction in scratching bouts over time incomparison to littermate controls (see e.g., FIG. 8C). Additionally,skin inflammation was markedly reduced as determined by ear thicknessmeasurements (see e.g., FIG. 8D) and histopathologic assessment (seee.g., FIG. 8E-F). Taken together, these studies demonstrate thatpreviously unrecognized sensory neuron-restricted IL-4Rα signaling iscritical for both the elicitation of chronic itch as well as thedevelopment of skin inflammation.

Example 6: Disruption of Neuronal JAK1 Signaling Reduces Chronic Itch

Given that type 2 cytokines are known to signal through JAK-dependentpathways in immune cells (Kelly-Welch et al., 2003; Schwartz et al.,2016), we sought to confirm that sensory neurons express JAK signalingcomponents at the single cell level. To examine this, we againreanalyzed a previously published data set of single-cell RNA-sequencingof DRG neurons (Usoskin et al., 2015) and identified that JAK1 is highlyexpressed in pruriceptive neurons (see e.g., FIG. 15). Thus, wehypothesized that JAK inhibition may represent a novel therapeuticstrategy for chronic itch. To test whether pharmacologic inhibition ofJAK signaling in vivo would reduce itch, we employed MC903 treatment toinduce AD-associated itch in WT mice while concurrently treating withthe JAK inhibitor ruxolitinib (see e.g., FIG. 9A). Indeed, systemicintraperitoneal (i.p.) delivery of ruxolitinib significantly reducedscratching bouts in MC903-treated mice (see e.g., FIG. 9B).Surprisingly, we observed that ear skin thickening (see e.g., FIG. 9C)and histologic parameters of disease (see e.g., FIG. 9D-E) demonstrateda statistically significant, but only slight, reduction in the settingof systemic ruxolitinib treatment despite its well-establishedanti-inflammatory properties. Thus, we speculated that the anti-itcheffects of ruxolitinib may be mediated predominantly through directneuronal JAK inhibition rather than through suppression of skininflammation.

To test whether JAK blockade in the neuronal compartment alone would besufficient to reduce chronic itch, we induced AD-like itch with MC903 inWT mice and delivered a single low dose of ruxolitinib intrathecally(i.t.) (see e.g., FIG. 9F). Strikingly, mice that received i.t.ruxolitinib demonstrated marked reduction in scratching bouts 24 hoursfollowing a single injection (see e.g., FIG. 9G), despite no effect onperipheral skin inflammation (see e.g., FIG. 9H-J). Thus, pharmacologicJAK blockade limited to the nervous system was sufficient to abate itch.Taken together, these studies demonstrate that JAK inhibition representsa viable therapeutic strategy for chronic itch. Further, theseobservations provoke the hypothesis that JAK signaling in sensoryneurons is a key mechanism by which chronic itch is mediatedindependently of skin inflammation.

To investigate whether sensory neuron-specific JAK signaling isnecessary for elicitation of chronic itch, we generated mice in whichJAK1 is conditionally deleted in sensory neurons (Na_(v)1.8-Cre⁺JAK1^(fl/fl), JAK1^(Δneuron)) given the robust expression of JAK1 inpruriceptive neurons (see e.g., FIG. 15). Upon induction of AD-like itch(see e.g., FIG. 9K), JAK1^(Δneuron) mice exhibited a marked reduction inchronic itch (see e.g., FIG. 9L) even in the presence of robust skininflammation (see e.g., FIG. 9M-O). Taken together, these studiesdemonstrate that neuronal JAK1 is critically important for theelicitation of chronic itch. As such, JAK1 is shown to be atherapeutically relevant target chronic itch disorders that presents inthe presence of an inflammatory etiology.

Example 7: Chronic Idiopathic Pruritus (CIP) Patients Exhibit SevereItch and Neuronal Dysregulation in the Absence of Overt SkinInflammation

Given the strong link between skin inflammation and itch in the settingof AD, we sought to study another itch disorder, chronic idiopathicpruritus (CIP), which presents in the absence of overt skin inflammationto broaden our understanding of chronic itch. CIP is strongly associatedwith aging and believed to be a manifestation of systemic immunesenescence (Norman, 2003; Patel and Yosipovitch, 2010; Reich et al.,2011). As a result, it has been proposed that CIP patients exhibit atype 2 immune profile due to loss of type 1 immunity (Berger andSteinhoff, 2011). In support of this, we recently showed that CIPpatients exhibit features of systemic type 2 inflammation associatedwith low-grade peripheral eosinophilia and elevation of IgE (Xu et al.,2016). Though successful elimination of skin inflammation in AD is knownto eliminate symptoms of itch, patients with CIP are often refractory toanti-inflammatory agents.

Unlike AD, a characteristic inflammatory skin disease (see e.g., FIG.10A), CIP presents with grossly normal skin findings (see e.g., FIG.10B). Further, AD histopathology demonstrates clear features of skininflammation including hyperkeratosis, acanthosis, and a robust mixeddermal inflammatory infiltrate (see e.g., FIG. 10C). However,histopathology of CIP patients exhibits only minimal inflammation uponbiopsy from even the most pruritic cutaneous sites, as evidenced by anormal epidermis (no acanthosis) with an overlying healthy “basketweave-patterned” stratum corneum (no hyperkeratosis) (see e.g., FIG.10D). Indeed, histologic grading of patient biopsies confirmed that CIPpatients exhibit markedly lower levels of skin inflammation compared toAD patients (see e.g., FIG. 10E). Remarkably, despite much milder skininflammation, CIP patients exhibit a higher Numerical Rating Scale (NRS)itch score compared to AD patients (AD NRS itch score: 5.55±0.51, CIPNRS itch score: 7.07±0.26) (see e.g., FIG. 10F). Taken together, thesefindings demonstrate that despite reduced skin inflammation incomparison to AD patients, CIP patients have more debilitating symptomsof itch. Thus, these findings show that chronic itch can manifest bothin the setting of robust skin inflammation as well as in the absence ofnotable inflammatory processes.

To characterize CIP at the molecular level, we performed RNA-sequencingon skin from four CIP patients, on lesional skin from four AD patients,and on healthy skin from four control subjects. Clustering of each groupby expression of the top 100 most differentially expressed genes betweencontrol and AD skin revealed that CIP skin is more closely related to ADskin than control skin (see e.g., FIG. 10G). These findings suggest thatCIP exhibits molecular signatures that are shared with AD despite markeddifferences in terms of skin inflammation. However, gene set enrichmentanalysis (GSEA) (Subramanian et al., 2005) comparing CIP and AD skinrevealed specific transcriptional programs that distinguish CIP from AD.This analysis showed that AD skin is associated with broad activation ofimmune responses (see e.g., FIG. 10H, TABLE 3) and that CIP skin isassociated with dysregulation of neuronal processes and sensoryperception (see e.g., FIG. 10H, TABLE 4). Collectively, these datademonstrate that, in contrast to the marked proinflammatory signature ofAD, CIP is a primary itch condition characterized predominantly by aprofile of neuronal dysfunction in association with minimalinflammation. As such, JAK1 is shown to be a therapeutically relevanttarget chronic itch disorders that presents in the absence of aninflammatory etiology or minimal inflammatory etiology.

TABLE 3 AD skin demonstrates enrichment of immune activation pathways.Relates to FIG. 10. List of top 50 biological process GO terms enrichedin AD in a direct comparison of CIP and AD as determined by gene setenrichment analysis (GSEA). Normalized enrichment score (NES) and falsediscovery rate (FDR) q-value of individual GO terms are provided. GOAccession FDR Number Name NES q-value GO:0002682 regulation of immunesystem process −8.77036 <10⁻⁵ GO:0002376 immune system process −8.66961<10⁻⁵ GO:0080134 regulation of response to stress −8.02582 <10⁻⁵GO:0050776 regulation of immune response −7.8129  <10⁻⁵ GO:0022613ribonucleoprotein complex biogenesis −7.7964  <10⁻⁵ GO:0002684 positiveregulation of immune system −7.77017 <10⁻⁵ process GO:0006952 defenseresponse −7.49978 <10⁻⁵ GO:0044403 symbiosis, encompassing mutualism−7.30644 <10⁻⁵ through parasitism GO:0050778 positive regulation ofimmune −7.20636 <10⁻⁵ response GO:0044419 interspecies interactionbetween −7.18966 <10⁻⁵ organisms GO:0042254 ribosome biogenesis −7.11062<10⁻⁵ GO:0031347 regulation of defense response −7.10497 <10⁻⁵GO:0048584 positive, regulation of response to −7.05507 <10⁻⁵ stimulusGO:0006396 RNA processing −7.04048 <10⁻⁵ GO:0009607 response to bioticstimulus −6.93027 <10⁻⁵ GO:0043604 amide biosynthetic process −6.89239<10⁻⁵ GO:0034660 ncRNA metabolic process −6.8318  <10⁻⁵ GO:0006955immune response −6.73395 <10⁻⁵ GO:0002253 activation of immune response−6.70878 <10⁻⁵ GO:0006518 peptide metabolic process −6.6791  <10⁻⁵GO:0016072 rRNA metabolic process −6.56767 <10⁻⁵ GO:1901564organonitrogen compound metabolic −6.55732 <10⁻⁵ process GO:0019058viral life cycle −6.53858 <10⁻⁵ GO:0031349 positive regulation ofdefense −6.52802 <10⁻⁵ response GO:0034470 ncRNA processing −6.35599<10⁻⁵ GO:1901566 organonitrogen compound −6.31783 <10⁻⁵ biosyntheticprocess GO:0043603 cellular amide metabolic process −6.24033 <10⁻⁵GO:0061024 membrane organization −6.19839 <10⁻⁵ GO:0032101 regulation ofresponse to external −6.11073 <10⁻⁵ stimulus GO:0045184 establishment ofprotein localization −6.09473 <10⁻⁵ GO:0071310 cellular response toorganic substance −6.08981 <10⁻⁵ GO:0002218 activation of innate immuneresponse −6.07281 <10⁻⁵ GO:0034097 response to cytokine −6.07209 <10⁻⁵GO:0009057 macromolecule catabolic process −6.07098 <10⁻⁵ GO:0045089positive regulation of innate immune −6.04607 <10⁻⁵ response GO:0051649establishment of localization in cell −6.03101 <10⁻⁵ GO:0007005mitochondrion organization −6.02195 <10⁻⁵ GO:0008104 proteinlocalization −6.0172  <10⁻⁵ GO:0045088 regulation of innate immuneresponse −5.9712  <10⁻⁵ GO:0065003 macromolecular complex assembly−5.95253 <10⁻⁵ GO:0071822 protein complex subunit organization −5.90784<10⁻⁵ GO:1902580 single-organism cellular localization −5.87975 <10⁻⁵GO:0070727 cellular macromolecule localization −5.83766 <10⁻⁵ GO:0010941regulation of cell death −5.80849 <10⁻⁵ GO:0031399 regulation of proteinmodification −5.80517 <10⁻⁵ process GO:0009617 response to bacterium−5.7918  <10⁻⁵ GO:0006413 translational initiation −5.75062 <10⁻⁵GO:0051247 positive regulation of protein −5.73842 <10⁻⁵ metabolicprocess GO:0009605 response to external stimulus −5.73487 <10⁻⁵

TABLE 4 CIP skin demonstrates enrichment of transcriptional programsassociated with neuronal processes and sensory perception. Relates toFIG. 10. List of top 50 biological process GO terms enriched in CIP in adirect comparison of CIP and AD as determined by gene set enrichmentanalysis (GSEA). Normalized enrichment score (NES) and false discoveryrate (FDR) q-value of individual GO terms are provided. GO Accession FDRq- Number Name NES value GO:0032990 cell part morphogenesis 3.0934610.002029 GO:0044782 cilium organization 2.879501 0.002522 GO:0060271cilium morphogenesis 2.867324 0.002018 GO:0008589 regulation ofsmoothened 2.766102 0.003024 signaling pathway GO:0016054 organic acidcatabolic process 2.669138 0.006637 GO:0046395 carboxylic acid catabolicprocess 2.666201 0.005615 GO:0051965 positive regulation of synapse2.584534 0.010122 assembly GO:0016339 calcium-dependent cell-cell2.545489 0.011865 adhesion via plasma membrane cell adhesion moleculesGO:0033539 fatty acid beta-oxidation using 2.528827 0.012158 acyl-CoAdehydrogenase GO:0010927 cellular component assembly 2.521315 0.011644involved in morphogenesis GO:0048667 cell morphogenesis involved in2.399559 0.028197 neuron differentiation GO:0072329 monocarboxylic acidcatabolic 2.391039 0.027353 process GO:0007156 homophilic cell adhesionvia 2.374797 0.027991 plasma membrane adhesion molecules GO:0097485neuron projection guidance 2.356602 0.029745 GO:0098742 cell-celladhesion via plasma- 2.32599  0.035653 membrane adhesion moleculesGO:0051298 centrosome duplication 2.27191  0.048845 GO:0009062 fattyacid catabolic process 2.266665 0.047421 GO:0061512 protein localizationto cilium 2.250689 0.050193 GO:0030030 cell projection organization2.228846 0.055343 GO:0003002 regionalization 2.225651 0.053408GO:0050807 regulation of synapse 2.176872 0.069665 organizationGO:0007224 smoothened signaling pathway 2.16341  0.072499 GO:0048812neuron projection 2.149574 0.076482 morphogenesis GO:0019228 neuronalaction potential 2.135246 0.080783 GO:0071236 cellular response toantibiotic 2.118108 0.08638  GO:0021915 neural tube development 2.1115970.086502 GO:0021522 spinal cord motor neuron 2.103877 0.087523differentiation GO:0007600 sensory perception 2.094911 0.089596GO:0007389 pattern specification process 2.093567 0.087319 GO:0050953sensory perception of light 2.082521 0.090076 stimulus GO:0021510 spinalcord development 2.050129 0.106422 GO:0000732 strand displacement2.047653 0.104586 GO:0045494 photoreceptor cell maintenance 2.0453060.103041 GO:0032989 cellular component 2.042259 0.10153  molphogenesisGO:0021515 cell differentiation in spinal cord 2.028853 0.107171GO:0019748 secondary metabolic process 2.026103 0.10577  GO:1903825organic acid transmembrane 2.017675 0.108687 transport GO:0035058nonmotile primary cilium 2.012586 0.108942 assembly GO:0060401 cytosoliccalcium ion transport 1.995952 0.117028 GO:0097553 calcium iontransmembrane 1.986171 0.121127 import into cytosol GO:0031023microtubule organizing center 1.977455 0.124149 organization GO:0007517muscle organ development 1.97117  0.125907 GO:1902656 calcium ion importinto cytosol 1.96663  0.123353 GO:0007098 centrosome cycle 1.96972 0.121292 GO:0086010 membrane depolarization during 1.964156 0.122664action potential GO:0098534 centriole assembly 1.960917 0.122407GO:0070509 calcium ion import 1.959427 0.121073 GO:0009953dorsal/ventral pattern formation 1.955245 0.121612 GO:0097237 cellularresponse to toxic 1.954725 0.119552 substance

Example 8: JAK Inhibition Improves Pruritus in Patients with CIP

Recent proof-of-concept pilot studies and Phase II clinical trials inpatients with atopic dermatitis (AD) have demonstrated rapid andsignificant reduction of itch symptoms in response to treatment with thecommercially available JAK inhibitor tofacitinib (Bissonnette et al.,2016; Levy et al., 2015). Whereas these studies sought to investigateJAK inhibition as an anti-inflammatory treatment for AD, our preclinicalfindings that both pharmacologic JAK inhibition (see e.g., FIG. 9A-J)and lineage-specific neuronal JAK1 deletion (see e.g., FIG. 9K-O) limititch suggest that JAK inhibition represents a novel neuromodulatoryapproach to selectively target itch. Therefore, despite the absence ofovert skin inflammation in CIP, we hypothesized that CIP patients maybenefit from JAK blockade.

Due to the lack of FDA-approved therapeutics specifically for theindication of CIP, we prescribed off-label treatment with the JAKinhibitor tofacitinib to five patients with severe CIP. All of thesepatients had previously failed multiple other off-label treatmentsincluding potent immunosuppression (TABLE 5). However, one monthfollowing treatment with oral tofacitinib, all five patientsdemonstrated marked improvement in their NRS itch scores (see e.g., FIG.11A). Strikingly, patients reported rapid onset of itch relief with JAKblockade despite previous immunosuppressive therapy (see e.g., FIG.11B). Thus, these findings suggest that JAK inhibition may represent anovel therapeutic strategy for patients with chronic itch disorders thatare resistant to conventional anti-inflammatory agents alone. Takentogether, our studies reveal that classical immune signaling pathwaysfunction in the sensory nervous system to mediate chronic itch. Althoughthere are currently no FDA-approved treatments specifically for chronicitch, our discovery of the significance of these immune signalingpathways in the sensory nervous system may promote the development ofnew therapies that target these shared pathways.

TABLE 5 Characteristics of CIP patients treated with tofacitinib.Relates to FIG. 11. Demographics and relevant medical history includingpreviously failed treatments of CIP patients given tofacitinib.Previously failed treatments Additional History of History of TopicalOral Immuno- Gaba- Anti- Anti- Photo- Patient Age Sex AD malignancysteroids steroids suppression^(a) pentin histamines depressents therapy1 79 M None None Yes Yes Yes Yes Yes Yes N/A 2 50 F None None Yes YesN/A N/A Yes N/A N/A 3 60 M None None Yes Yes N/A N/A Yes N/A Yes 4 79 MNone Yes Yes Yes Yes Yes N/A Yes N/A 5 75 F None Yes Yes Yes N/A N/A N/AN/A Yes

Discussion

Chronic itch is a common example of pathologic dysregulation of sensoryresponses that are normally employed by mammals to remove invadingpathogens. It is a highly debilitating symptom of multiple dermatologic,neurologic, and systemic medical conditions. Moreover, chronic itch canalso present in the absence of a clearly defined disorder as in CIP(Berger and Steinhoff, 2011; Mollanazar et al., 2016; Xu et al., 2016;Yosipovitch and Bernhard, 2013). Although itch is an often overlookedsymptom in patient care, clinical studies have established that chronicitch has a profoundly negative impact on quality of life (Kini S P etal., 2011; Matterne et al., 2011; Ständer et al., 2007; Yosipovitch andBernhard, 2013). However, despite its substantial burden on society,there are currently no specific treatments indicated for chronic itch.

In the setting of AD, the type 2 cytokines IL-4, IL-5, and IL-13coordinate a complex inflammatory response that is critical for diseasepathogenesis. This inflammatory cascade leads to debilitating chronicitch. However, the mechanisms by which proinflammatory mediators, suchas the type 2 cytokines, elicit itch remain poorly understood. Asdescribed herein, three conceptual advances that broaden ourunderstanding of chronic itch were provided. First, it was demonstratedthat the classical immune signaling molecules IL-4 and IL-13 directlyactivate itch-sensory neurons and that sensory neuron-specific deletionof IL-4Rα is sufficient to abate chronic itch in vivo. Second, we showthat sensory neuron-specific deletion of JAK1, a key downstreamsignaling molecule for type 2 cytokines in immune cells, reduces chronicitch. Third, we demonstrate that, regardless of the level of skininflammation, pharmacologic JAK inhibition is sufficient to abate itch.Importantly, we show that patients with even idiopathic forms of chronicitch who are recalcitrant to standard immunosuppressive therapy improveon systemic JAK inhibitors. Taken together, the current study identifiesnovel functions of classical immune signaling pathways that regulatesensory perception.

Classically, the dominant source of type 2 cytokines in AD lesional skinwas believed to be the adaptive immune system via T helper type 2 (Th2)cells (Weidinger and Novak, 2016). Further, recent studies have shownthat Th2 cell-derived IL-31 can elicit scratching in vivo (Cevikbas etal., 2014; Dillon et al., 2004; Sonkoly et al., 2006). Thus, bothAD-associated inflammation and itch were largely believed to be mediatedby the adaptive immune system. However, we and others have recentlyshown that innate immune cell populations such as group 2 innatelymphoid cells (ILC2s) and basophils critically mediate skininflammation independently of the adaptive immune system (Imai et al.,2013; Kim et al., 2013a, 2014; Roediger et al., 2013; Salimi et al.,2013). In support of this, multiple studies have demonstrated that bothILC2s and basophils are critical sources of type 2 cytokines in vivo(Imai et al., 2013; Kim et al., 2013a, 2014; Roediger et al., 2013;Salimi et al., 2013). Based on our new data demonstrating that neuronalIL-4/13 signaling critically mediates chronic itch, we speculate that,in addition to adaptive Th2 cells, innate immune cells such as ILC2s andbasophils play critical roles in mediating itch. Future studies will beperformed to fully define the functional contributions of both adaptiveand innate immune cells to the development of chronic itch.

Upstream of the type 2 cytokine-associated immune response, thepredominately epithelial cell-derived cytokines thymic stromallymphopoietin (TSLP) and IL-33 have been shown to be master initiatorsof type 2 inflammation via their effects on a variety of cells includingTh2 cells as well as ILC2s and basophils (Hammad and Lambrecht, 2015;Kim and Artis, 2015; Kim et al., 2013b; Oetjen et al., 2016; Siracusa etal., 2013a). Strikingly, two recent studies have demonstrated that TSLPand IL-33 can act directly on sensory neurons and function aspruritogens (Liu et al., 2016; Wilson et al., 2013). Indeed, we andothers have shown that TSLP and IL-33 are highly expressed in thesetting of MC903-induced AD-like disease (Kim et al., 2013a; Li et al.,2006, 2009; Salimi et al., 2013). Despite high expression of thesecytokines and robust activation of numerous downstream cytokine pathwaysin AD, our study identifies that sensory neuron-specific deletion ofIL-4Rα alone is sufficient to abate itch. Thus, it appears that neuronalIL-4Rα signaling is an exquisitely robust target for AD-associated itch.However, whether IL-4Rα represents a novel itch-sensory pathway ormodulates the responsiveness of sensory neurons to previously identifiedpruritogens such as IL-31, TSLP, and IL-33 remains to be determined.

In addition to transmission of itch sensation, the marked reduction ofdisease severity in IL-4Rα^(Δneuron) mice provokes the hypothesis thatneuronal activation by type 2 cytokines may also mediate neurogenicinflammation. Strikingly, clinical reports have described improvementsin both AD and psoriasis lesions after denervation of the lesional skin,suggesting neurogenic components of both diseases (Amon and Wolff, 1994;Azimi et al., 2015; Raychaudhuri and Farber, 1993). In support of thispossibility, recent studies have shown that sensory neurons in the skincan promote Th17-associated inflammation and psoriasis (Kashem et al.,2015; Riol-Blanco et al., 2014). Whether neurogenic processes canregulate type 2 inflammation in the skin remains to be definitivelydetermined. In the lung, Talbot et al. have recently shown that the type2 cytokine IL-5 stimulates neurons projecting from the nodose ganglia tomediate allergic airway inflammation via release of vasoactiveintestinal peptide (VIP) (Talbot et al., 2015). However, we were notable to find evidence that IL-5 activates sensory neurons that innervatethe skin. Thus, the mechanisms and functional consequences of sensoryneuron activation by type 2 inflammation may be tissue-specific. Futurestudies will be required to determine what paradigms apply in the skinand the molecular mechanisms that underlie neurogenic type 2 skininflammation.

Phase II and III clinical trials for dupilumab, an anti-IL-4Rαmonoclonal antibody, have repeatedly demonstrated remarkable improvementin itch symptoms in patients (Beck et al., 2014; Simpson et al., 2016;Thaçi et al., 2016). Further, early clinical studies employing JAKinhibitors have demonstrated significant improvement of itch in patientswith AD (Bissonnette et al., 2016; Levy et al., 2015). Until now, thesechanges have been ascribed to the anti-inflammatory properties of IL-4Rαand JAK blockade. However, our study suggests that such improvements ofitch symptoms may occur independently of specific anti-inflammatoryproperties of these therapies and rather be attributable to theirpreviously unrecognized neuromodulatory effects. Based on the potentneuromodulatory effects of JAK inhibition and conditional geneticdeletion in mice, we hypothesized that JAK inhibition would work as ananti-itch therapy even in the absence of robust skin inflammation. Insupport of this, we found that patients with CIP, which presents withoutovert skin inflammation, improved significantly when treated withtofacitinib. Taken together, these findings suggest that JAK inhibitioncan serve dual anti-inflammatory and neuromodulatory roles. Importantly,our current studies have allowed us to design a prospective clinicaltrial exploring the use of a novel JAK1-selective inhibitor (INCB039110)in patients with CIP (clinicaltrials.gov ID: NCT02909569). However,larger prospective randomized-controlled trials will be required tofully determine the efficacy of such treatments.

Immunity at epithelial barrier surfaces is believed to have evolved tomediate rapid expulsion of pathogens as well as potential toxins.Whereas the epithelium was previously thought to be solely a physicalbarrier, recent advances have highlighted its direct role in a varietyof immune functions (Artis and Spits, 2015; Hammad and Lambrecht, 2015;Palm et al., 2012). In addition to the immune system and the epithelialbarrier itself, the mammalian host also employs behavioral responsessuch as coughing and scratching to mechanically remove noxious stimuliprior to tissue damage. Our current study demonstrates that classicalimmune mediators play an essential part in the sensory circuit tomodulate host behavior. By identifying previously unrecognizedneuroimmunologic pathways, we have uncovered novel therapeuticapproaches for the treatment of pathologic sensory disorders such aschronic itch.

Example 9: Methods

The following example describes the methods used in the previousExamples 2-8 unless expressed otherwise.

Mice

All mice were housed in standard environmental conditions (12 hourlight-dark cycle; 23° C.; food and water ad libitum) at WashingtonUniversity School of Medicine. C57Bl/6 and Rosa26-tdTomato mice werepurchased from The Jackson Laboratory. Na_(v)1.8-Cre mice were providedby Dr. Rohini Kuner (Heidelberg University). IL-4-eGFP (4get) mice wereprovided by Dr. Edward Pearce (MPI-IE). Il4ra^(flox) mice were providedby Dr. Ajay Chawla (UCSF). JAK1^(flox) mice were purchased from NanjingBiomedical Research Institute of Nanjing University. Na_(v)1.8-tdTomato⁺IL-4-eGFP⁺ reporter mice were generated by first crossing Na_(v)1.8-Cre⁺and Rosa26-tdTomato⁺ mice and then crossing the progeny to theIL-4-eGFP⁺ mice. IL-4Rα^(Δneuron) (Na_(v)1.8-Cre⁺ Il4ra^(fl/fl)) micewere generated by crossing the Na_(v)1.8-Cre⁺ and Il4ra^(flox) mice.JAK1^(Δneuron) (Na_(v)1.8-Cre⁺ JAK1^(fl/fl)) mice were generated bycrossing the Na_(v)1.8-Cre⁺ and Jak1^(flox) mice. Genotyping of mice wasperformed using standard PCR. All murine work was performed inaccordance with the guidelines of the Washington University School ofMedicine Department of Comparative Medicine Animal Care and UseCommittee (Protocol #20140170).

RNA Isolation from Murine DRG and TG and RT-qPCR

For RNA isolation, murine dorsal root ganglia (DRG) or trigeminalganglia (TG) were harvested, cleaned of connective tissues, andhomogenized in 1 mL Trizol Reagent (Life Technologies). Total RNA wasextracted with the RNeasy Mini Kit (Qiagen) following the manufacturer'sinstructions. Following extraction, samples were treated with DNase(Turbo DNA-Free Kit, Thermo Scientific) following the manufacturer'sinstructions. Next, cDNA was synthesized using the High-Capacity cDNAReverse Transcription Kit (Applied Biosystems). Gene expression levelswere determined by RT-qPCR (StepOnePlus; Applied Biosystems). Briefly,gene expression was normalized to Gapdh and relative expression wascalculated using either the ΔC_(t) or ΔΔC_(t) method where appropriate.For gel electrophoresis, products from RT-qPCR reactions were loadedonto a 2% agarose gel with ethidium bromide (1 μg/mL) and run at 140 Vfor 25 minutes. Primer and probe sequences used for each gene wereselected from pre-validated PrimeTime qPCR Assays (Integrated DNATechnologies). Primer and probe sequences used were:

1. Gapdh - Probe: (SEQ ID NO: 1)/56-FAM/TGCAAATGG/ZEN/CAGCCCTGGTG/3IABkFQ/ Primer 1: (SEQ ID NO: 2)GTGGAGTCATACTGGAACATGTAG Primer 2: (SEQ ID NO: 3) AATGGTGAAGGTCGGTGTG2. Il4ra - Probe: (SEQ ID NO: 4)/56-FAM/CAGAACCAG/ZEN/CAAGCACGCAGA/3IABkFQ/ Primer 1: (SEQ ID NO: 5)GTTACAGGAACAAGACCAGCA Primer 2: (SEQ ID NO: 6) TGGAGCCTGAACTCGCA3. Il5ra - Probe: (SEQ ID NO: 7)/56-FAM/TGAGCAAGC/ZEN/TTCTCCCACTGAGC/3IABkFQ/ Primer 1: (SEQ ID NO: 8)AGAATTAGTAACACAGGCACCA Primer 2: (SEQ ID NO: 9) CAAGGATCTAACCAGGGTCTTC4. Il13ra1 - Probe: (SEQ ID NO: 10)/56-FAM/ACAGGTGGC/ZEN/TGAACTTCTGTGGC/3IABkFQ/ Primer 1: (SEQ ID NO: 11)GAGATTTTCGACAGAGACGCT Primer 2: (SEQ ID NO: 12) CTGTTGGTGCTGCTACTGT5. Il31ra - Probe: (SEQ ID NO: 13)/56-FAM/TCGTCATCT/ZEN/GAGAGGCCATAAACAACTC/3IABkFQ/ Primer 1:(SEQ ID NO: 14) GATCGTCTGCTTCTCTTACACC Primer 2: (SEQ ID NO: 15)TAGTGCCGTTCTGTGATCAG

RNA Isolation from Human DRG and RT-PCR

Human dorsal root ganglia (hDRG) were acquired from de-identified UStransplant donors under an IRB-exempt protocol. After hDRG extraction,fat, dura, and connective tissues were removed as previously described(Valtcheva et al., 2016). The hDRG were subsequently stored in RNAlater(Life Technologies) at −80° C. until RNA isolation was performed. ForRNA isolation, tissue from one half of one hDRG was homogenized in 1 mLTrizol Reagent (Life Technologies) following the manufacturer'sinstructions. Following total RNA extraction, genomic DNA was eliminatedand cDNA synthesized using the Maxima H Minus First Strand cDNASynthesis Kit with dsDNase (Thermo Scientific) following themanufacturer's instructions. RT-PCR product was loaded onto a 2% agarosegel with ethidium bromide (1 μg/mL) and run at 100 V for 45 minutes.RT-PCR was performed using the following primer sets:

1. IL4RA - Primer 1: GACGTGGTCAGTGCGGATAA (SEQ ID NO: 16) Primer 2:CTGAAATCTGCCGGGTCGTT (SEQ ID NO: 17) 2. IL5RA - Primer 1:CTTGCGGTGCTTGTTAACGG (SEQ ID NO: 18) Primer 2: CGAGTGAACGGGTACGTTTCT(SEQ ID NO: 19) 3. IL13RA1 - Primer 1: CCTACGGAAACTCAGCCACC(SEQ ID NO: 20) Primer 2: CGAGTGAACGGGTACGTTTCT (SEQ ID NO: 21)4. IL31RA - Primer 1: CACAAGAAAAGCTCGCAGACA (SEQ ID NO: 22) Primer 2:GGTGGTTCAGTTTTCGCTATGTT (SEQ ID NO: 23)

Overnight Murine DRG Neuron Culture

Murine DRG neurons were isolated and cultured using a previouslypublished protocol with slight modification (Malin et al., 2007).Briefly, laminectomies were performed on mice and bilateral DRG wereremoved. After removal of connective tissues, DRG were transferred to 1mL Ca²⁺/Mg²⁺-free Hank's Balanced Salt Solution (HBSS) containing 1 μLsaturated NaHCO₃, 0.35 mg L-cysteine, and 20 U papain (Worthington) andincubated at 37° C. for 20 min. The suspension was centrifuged, and thesupernatant was removed and replaced with 1 mL Ca²⁺/Mg²⁺-free HBSScontaining 3.75 mg collagenase type II (Worthington) and 7.5 mg dispase(Worthington) and incubated at 37° C. for 20 min. After digestion,neurons were gently triturated, pelleted, and then resuspended inNeurobasal-A medium containing 2% B-27 supplement (Gibco), 100 U/mLpenicillin plus 100 μg/mL streptomycin (Sigma), 100 ng/mL nerve growthfactor (Sigma), and 10% heat-inactivated FBS (Sigma). Neurons were thenplated on a 12 mm glass coverslip pre-coated with poly-L-lysine (Sigma)and laminin (Sigma) and cultured under a humidified atmosphere of 5% CO₂at 37° C. for 18-24 hours before use.

Calcium Imaging

Cultured DRG neurons were loaded with 4 μM Fura-2 AM (Life Technologies)in overnight DRG culture medium at 37° C. for 45 minutes. Cells werethen washed three times and incubated in calcium imaging buffer (130 mMNaCl, 3 mM KCl, 2.5 mM CaCl₂, 0.6 mM MgCl₂, 10 mM HEPES, 10 mM glucose,1.2 mM NaHCO₃, pH 7.45) at room temperature for 30 minutes before use.All recombinant murine cytokines (IL-4, IL-5, and IL-13: Peprotech,IL-31: Bristol-Myers Squibb) were used at 300 nM, while histamine(Sigma) and capsaicin (Sigma) were used at 50 μM and 300 nM,respectively. Only sensory neurons that responded to a final challengeof 100 mM KCl were used in analyses. Fluorescence was recorded at 340 nmand 380 nm excitation wavelengths (F340, F380) using an inverted NikonTi-E microscope with NIS-Elements imaging software (Nikon Instruments).Fluorescence ratios (F340/F380) were normalized to baseline and used toreflect changes in intracellular Ca²⁺ and neuronal activation uponstimulation. Cells were considered responsive if they demonstrated achange in fluorescence ratio >10% of baseline. Data were derived fromtwo to three independent experiments.

MC903 Treatment and Pathology Assessment

For induction of AD-like inflammation and itch, mice were topicallytreated once daily with 2 nmol of MC903 (calcipotriol, TocrisBioscience) for 7 days or 1 nmol of MC903 for 12 days on both ears in 20μL of ethanol (vehicle) as previously described (Kim et al., 2013a,2014, Siracusa et al., 2011, 2013b). Ear thickness measurements wereperformed daily with dial calipers as previously described (Kim et al.,2013a, 2014). At the end of the MC903 treatment, murine ear skin tissueswere fixed in 4% paraformaldehyde (PFA) and embedded in paraffin beforesectioning and staining with Hematoxylin & Eosin (H&E). Histology scorewas determined by the following formula using ImageJ analysis software(NIH) as previously described: (total number of lymphocytes per highpower field (HPF)+thickness of the epidermis measured in microns fromthe basement membrane to the top of the stratum corneum) divided by 100(Kim et al., 2014). All images were captured with a NanoZoomer 2.0-HTSystem (Hamamatsu). Murine disease and behavioral assessment data(below) were derived from two to three independent experiments.

Behavioral Assessment

For behavioral assessments, mice were first acclimated to the recordingroom and behavior chambers at least one day prior to testing. On the dayof testing, mice were again acclimated to the recording room andbehavior chambers and then video recorded. The video recordings werethen manually scored for number of scratching bouts per 30 minute block.One scratching bout was defined as one instance of lifting the hind pawfrom the floor, scratching, and returning the paw to the floor orplacing the paw in the animal's mouth. All behavioral tests wereperformed on sex- and age-matched adult mice (8-12 weeks old).

Murine and Human Skin RNA Extraction

Samples of murine skin were obtained and stored in RNAlater (Sigma) at−80° C. before processing following the manufacturer's instructions.Human skin tissues were obtained as 4-mm punch biopsies and stored inRNAlater (Sigma) at −80° C. before processing. To extract whole tissueRNA, samples were homogenized using a bead homogenizer and processedusing the Qiagen RNeasy kit following the manufacturer's instructions.Following total RNA extraction, samples were treated with DNase (TurboDNA-Free Kit, Thermo Scientific) following the manufacturer'sinstructions before sequencing library preparation. All human skinsamples for RNA-sequencing analysis were acquired under protocolsapproved by the Washington University in St. Louis IRB (Protocols#201410014, #201412117, and #201507042).

Library Preparation and RNA-Sequencing

Library preparation, sequence alignment, and determination of transcriptabundance were carried out by the Genome Technology Access Center (GTAC)at Washington University School of Medicine. Briefly, librarypreparation was performed with 1 ug of total RNA, and RNA integrity wasdetermined using an Agilent Bioanalyzer. Ribosomal RNA was removed by ahybridization method using Ribo-ZERO kits (Illumina-EpiCentre). mRNA wasthen fragmented in buffer containing 40 mM Tris Acetate (pH 8.2), 100 mMPotassium Acetate, and 30 mM Magnesium Acetate and heated to 94° C. for150 seconds. mRNA was reverse transcribed to yield cDNA usingSuperScript III RT enzyme (Life Technologies, per manufacturer'sinstructions) and random hexamers. A second strand reaction wasperformed to yield ds-cDNA. cDNA was blunt ended, had an A base added tothe 3′ ends, and then had Illumina sequencing adapters ligated to theends. Ligated fragments were then amplified for 12 cycles using primersincorporating unique index tags. Fragments were sequenced on an IlluminaHiSeq-3000 using single reads extending 50 bases.

RNA-sequencing reads were aligned to the Ensembl release 76 assemblywith STAR version 2.0.4b. Gene counts were derived from the number ofuniquely aligned unambiguous reads by Subread:featureCount version1.4.5. Transcript counts were produced by Sailfish version 0.6.3.Sequencing performance was assessed for total number of aligned reads,total number of uniquely aligned reads, genes and transcripts detected,ribosomal fraction, known junction saturation, and read distributionover known gene models with RSeQC version 2.3.

Differential Gene Expression and Gene Set Enrichment Analysis

Differential gene expression analysis and sample clustering fromRNA-sequencing data were performed with the DESeq2 package available inR using default configurations. Genes with a two-fold or greater foldchange and an adjusted p-value <0.1 were considered significant.Dendrograms in heat maps were formed by hierarchical clustering on theEuclidean distances between samples. Gene set enrichment analysis (GSEA)was carried out using the preranked gene list feature available in thejavaGSEA application available online(http://www.broadinstitute.org/gsea/downloads.jsp). Genes were prerankedby the product of the fold change and the inverse of the adjustedp-value of differential expression as determined by DESeq2. Gene setswere derived from the Biological Process Ontology.

Intravital Two-Photon Imaging

Time-lapse imaging was performed with a custom built two-photonmicroscope equipped with a 1.0 NA 20× water dipping objective (Olympus)running ImageWarp acquisition software (A&B Software) as previouslydescribed (Zinselmeyer et al., 2009). In brief, mice were anesthetizedand VetBond (3M) was used to attach the ear to a plastic cover glassthat was secured to a heated support. Mice were placed on a warming padand given intraperitoneal (i.p.) saline for hydration for imagingexperiments lasting more than 90 minutes. A drop of water was placed onthe skin and the ears were imaged directly with the water dippingobjective. Video-rate imaging was used to identify sites withNa_(v)1.8-tdTomato⁺ nerve fibers. IL-4-eGFP⁺ cell migration dynamicswere analyzed using 3D time-lapse imaging for up to 3 hours for eachtime point. Tissue was imaged with a Chameleon Vision II Ti:Sapphirelaser (Coherent) tuned to 915 nm and fluorescence emission detected byPMTs simultaneously using 495 nm and 560 nm dichroic filters: Blue (<495nm, SHG collagen), green (495-560 nm, eGFP) and red (>560 nm, tdTomato).Autofluorescence of the epidermis appears as mix of color (495-600 nm)and thus can be discriminated from IL-4-eGFP⁺ cells. For time-lapseimaging, we acquired a 220×240×75 μm volume as 31 sequential 2.5 μmz-steps with a time resolution of approximately 42 seconds. X,Y wasresolution 0.75 μm/pixel, which is adequate to resolve individualIL-4-eGFP⁺ cells and nerve fibers. Multi-dimensional data sets wererendered in 3D using Imaris (Bitplane). Cell tracking and data analysiswere performed using Imaris and Motility Lab (2ptrack.net). IL-4-eGFP⁺cells were defined as sensory fiber-associated if they overlapped withsensory fibers and the contacts between IL-4-eGFP⁺ cells andNa_(v)1.8-tdTomato⁺ nerve fibers were confirmed manually in 3D images.

Pharmacologic JAK Inhibition

Stock ruxolitinib (Selleck Chemicals) solutions were prepared in DMSO(Sigma) per the manufacturer's instructions. For systemic treatments,mice treated twice daily with 100 μg ruxolitinib diluted in 100 μl PBSby intraperitoneal (i.p.) injection. For intrathecal (i.t.) ruxolitinibtreatments, mice were injected 24 hours before behavioral assays with 10μg ruxolitinib diluted in 10 μl PBS. Control mice were injected withappropriate vehicle controls (DMSO in PBS) of equivalent volumes.

Human Histopathology

Human skin tissues were obtained as 4-mm punch biopsy specimens thenfixed in 10% paraformaldehyde (PFA) and embedded in paraffin. Sectionswere stained with H&E. Clinical analyses of patient biopsies werecarried out by the Washington University Dermatopathology Center. Allsamples were collected from fully consented individuals, and humanstudies were approved by the Washington University in St. Louis IRB(Protocols #201410014 and #201412117). Histology score was determined bythe following formula using ImageJ analysis software (NIH) as previouslydescribed: (total number of lymphocytes per high power field(HPF)+thickness of the epidermis measured in microns from the basementmembrane to the top of the stratum corneum) divided by 100 (Kim et al.,2014).

Numerical Rating Scale (NRS) Itch Scoring

Patients in the specialty Itch Clinic at the Center for AdvancedMedicine (CAM) at Washington University School of Medicine are routinelyassessed prospectively for their NRS itch score. Briefly, patients wereasked to rate their itch intensity over the past 24 hours from 0 to 10where 0 represents “no itch” and 10 represents the “worst itchimaginable”. For AD and CIP patient comparisons, NRS itch scores wereobtained by retrospective analysis of charts from patients seen betweenAugust 2014 and February 2016. For patients who received treatment withtofacitinib 5 mg by mouth twice daily, NRS itch scores were obtainedprospectively from the beginning of treatment. Recordings of daily NRSitch scores of two patients are reported in FIG. 7B during tofacitinibtreatment. These analyses were approved by the Washington University inSt. Louis IRB (Protocol #201605144).

Statistical Analysis

Data are presented as mean±SEM unless indicated otherwise. Statisticalsignificance was determined by unpaired Student's t-test with Welch'scorrection unless noted otherwise. Statistical analyses were performedusing Prism GraphPad software v7.0. Significance is labeled as:***p<0.001, **p<0.01, *p<0.05, N.S., Not Significant.

What is claimed is:
 1. A method for treating pruritus in a subject inneed thereof, the method comprising administering to the subject atherapeutically effective amount of a JAK1 inhibitor.
 2. The method ofclaim 1, wherein the subject is diagnosed with pruritus or chronicidiopathic pruritus and treatment prevents or reduces pruritus in thesubject.
 3. The method of claim 1, wherein the subject has extremelysevere itching or severe itching; or the subject has moderate or milditching.
 4. The method of claim 1, wherein the pruritus has lasted forat least at least seven weeks; the pruritus has lasted for at least atleast eight weeks; the pruritus has lasted for at least at least nineweeks; or the pruritus has lasted for at least at least ten weeks. 5.The method of claim 1, wherein treatment reduces severity of itching ina subject, increases number of itch-free days in the subject, improvesquality of life of the subject, or any combination thereof.
 6. Themethod of claim 1, wherein the subject is predisposed to pruritus orchronic idiopathic pruritus and treatment with the JAK inhibitorprevents a reoccurrence of chronic pruritus in the subject or reducesfrequency of acute pruritus in the subject.
 7. The method of claim 1,wherein the JAK inhibitor is selected from one or more of the groupconsisting of: tofacitinib, ruxolitinib, baricitinib, itacitnib(INCB039110), oclacitinib, AZD1480, fedratinib, AT9283, AG-490,momelotinib, TG101209, gandotinib, NVP-BSK805, AZ 960, CEP-33779,Pacritinib, XL019, S-Ruxolitinib, ZM 39923, Decernotinib, Cerdulatinib,filgotinib, FLLL32, BMS-911543, peficitinib, GLPG0634, GLPG0634analogue, Go6976, curcumol, cucurbitacin, lestaurtinib, upadacitinib,CHZ868, Solcitinib (GSK 2586184), NS-018; or a derivative thereof; orpharmaceutically acceptable salt thereof.
 8. The method of claim 1,wherein the JAK inhibitor is

or a combination thereof.
 9. The method of claim 1, where the JAKinhibitor is administered parenterally, intrathecally, intranasally, ororally.
 10. The method of claim 1, wherein the JAK inhibitor isadministered on a daily basis, administered on a daily basis for atleast 7 consecutive days, at least 14 consecutive days, or at least 30consecutive days.
 11. The method of claim 1, wherein the pruritus is asymptom of broad activation of immune responses or dysregulation ofneuronal processes and sensory perception.
 12. The method of claim 1,wherein the subject is diagnosed with, or the pruritus is a symptom of,a disease or condition selected from one or more of the group consistingof: allergic reaction, arthropod bites, athlete's foot, atopicdermatitis (AD), atopic itch, atopic dermatitis-associated itch,autoimmune connective tissue disease, bacterial infection, biliary itch,broad activation of immune responses, body louse, bullous diseases,brachioradial pruritus, brain tumors, chronic idiopathic pruritus,contact dermatitis, cholestasis, cutaneous larva migrans, cutaneousT-cell lymphoma, nervous system damage, dandruff, delusionalparasitosis, dermatomyositis, dermatosis of pregnancy, diabetesmellitus, drug eruptions, dysregulation of neuronal processes andsensory perception, eczema, eosinophilic folliculitis, foreign objectsor devices on skin, fungal infection, gestational pemphigoid, head lice,herpes, hidradenitis suppurativa, hives, Hodgkin's disease,hyperparathyroidism, idiopathic chronic itch, inflammation, insectinfestation, insect bites, insect stings, intrahepatic cholestasis ofpregnancy, iron deficiency anemia, increased accumulation of exogenousopioids or synthetic opioids, internal cancer, jaundice, lichen planus,lichen sclerosus, lupus erythematosus, lymphoma, lymphoma-associateditch, leukemia-associated itch, malignancy, mastocytosis, menopause,multiple sclerosis, neoplasm, nerve irritation, neurogenic itch,neuropathic itch, notalgia paresthetica, notalgia obsessive-compulsivedisorders, paresthetica, parasitic infection, papular urtcaria,pediculosis, peripheral neuropathy, photodermatitis, polycythemia vera,psychiatric disease, psychogenic itch, pruritic popular eruption of HIV,pruritic urticarial papules and plaques of pregnancy (PUPPP), psoriasis,psoriasis-associated itch, psoriatic itch, pubic lice, punctatepalmoplantar keratoderma, renal itch, rheumatoid arthritis, scabies,scar growth, shaving, seborrheic dermatitis, stasis dermatitis, sunburn,swimmer's itch, systemic immune senescence, tactile hallucinations,Th17-associated inflammation, thyroid illness, uraemia, pruritus oruremic itch, urticaria, urticarial itch, varicella, viral infection,wound or scab healing, and xerosis.
 13. The method of claim 1, whereinthe pruritus has an inflammatory etiology; the pruritus has neuronaldysfunction etiology; or the pruritus has an unknown etiology.
 14. Themethod of claim 1, wherein the JAK inhibitor is a TRPV1 inhibitor;modulates signaling of IL-4 or IL-13 pathway; or targets an IL-4Rasignaling pathway.
 15. The method of claim 1, further comprisingadministration of a TRPV1 inhibitor, dupilumab, or secukinumab.
 16. Themethod of claim 1, wherein the pruritus is chronic idiopathic pruritus.17. The method of claim 16, wherein the JAK inhibitor is selected fromTofacitinib or Ruxolitinib or a combination thereof.
 18. The method ofclaim 16, wherein the JAK inhibitor is not oclacitinib.
 19. A method oftreating chronic idiopathic pruritus in a human subject comprisingadministering to the subject a therapeutically effective amount of a JAKinhibitor selected from the group consisting of Tofacitinib andRuxolitinib or a combination thereof.
 20. The method of claim 1, whereinthe pruritus is not associated with atopic dermatitis.
 21. The method ofclaim 1, wherein the pruritus comprises a neurogenic itch component.